Electrochemical system and implantable biochemical test chip

ABSTRACT

The present disclosure provides an electrochemical system, including an electrode unit and a reactive unit electrically coupled to the electrode unit. The electrode unit includes a working electrode and a counter electrode, wherein a current density of the counter electrode is greater than a current density of the working electrode. An implantable biochemical test chip is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefits of prior-filed U.S. provisionalapplication No. 63/343,294, filed on May 18, 2022. The entire contentsof all of which are hereby incorporated by reference.

TECHNICAL FIELD

The present application relates to an electrochemical system and animplantable biochemical test chip for use in medical measurement,particularly to an electrochemical system and an implantable biochemicaltest chip capable of increasing an electrical neutrality of a counterelectrode.

BACKGROUND

In-vitro medical measurement plays a vital role in today's medicalindustry; by qualitatively and quantitatively measuring biologicalfluids changes, it provides index information for rapid diagnosis andtreatment of diseases. The use of biochemical test chips has become astandard technique for medical or biochemical testing.

Conventional biochemical test chips have at least two electrodes. Afterloading the specimen into the biochemical test chip's reaction zone, theelectrochemical properties of the specimen can be measured using saidtwo electrodes. When the specimen undergoes an electrochemical reaction,a change in current occurs, and this current change is linearlyproportional to the concentration of substances in the specimen that canundergo the redox reaction. Therefore, one can analyze the specimen'sconcentration by measuring the current generated by the redox reactionon the electrode surface.

In recent years, as the need to reduce the sampling volume hasincreased, one technology trend is to reduce the size of the electrodesof the biochemical test chip to reduce the amount of specimen required.However, the reduction in the size of the electrode results in low orweak electrochemical signals. Therefore, biochemical test chips areoften additionally disposed with a conductive medium to enhance themeasurement signal. However, adding the conductive medium will increasethe manufacturing cost and the difficulty in dissolving and drying.Moreover, when measuring specimens with high concentrations, abottleneck effect may occur when the flow of electrons reacting to theworking electrode exceeds the total amount that can be reacted with thecounter electrode, thereby limiting the range of concentrations that canbe measured by the biochemical test chip.

The “prior art” discussion above merely provides a technology backgroundwithout acknowledging that the “prior art” discussed above reveal thesubject matter of this disclosure and do not constitute prior art atthis time, and that any of the “prior art” discussion above should notbe regarded as any part of the present application.

SUMMARY OF THE INVENTION

The present disclosure provides a biochemical test chip, including aninsulating substrate, an electrode unit, a first insulating septum, areactive layer, and a second insulating septum. The electrode unit islocated on the insulating substrate. The electrode unit includes aworking electrode and a counter electrode, wherein the current densityof the counter electrode is greater than the current density of theworking electrode. The first insulating septum is located on theelectrode unit. The first insulating septum has a first opening, whereinthe first opening at least partially exposes the electrode unit. Thereactive layer is located on the first opening and is electricallyconnected to the electrode unit. The second insulating septum is locatedon the first insulating septum.

In some embodiments, the current density of the counter electrode isgreater than or equal to density of the working electrode.

In some embodiments, the area of the counter electrode is smaller thanor equal to the area of the working electrode.

In some embodiments, the reactive layer and a target analyte undergo aprimary reaction, and the counter electrode is configured to undergo asecondary reaction, wherein the secondary reaction does not interferewith the primary reaction, and the secondary reaction allows the counterelectrode to have the capability to receive or release additionalelectrons.

In some embodiments, the counter electrode includes a first portion anda second portion, wherein the first portion and the reactive layer donot overlap with each other.

In some embodiments, the counter electrode includes a first portion anda second portion, wherein the opening at least partially exposes thefirst portion.

In some embodiments, the biochemical test chip further includes aprotective layer, which is electrically connected to the electrode unit.

In some embodiments, the electrode unit further includes a secondcounter electrode, wherein the counter electrode and the second counterelectrode are separated from each other.

In some embodiments, a standard reduction potential of the counterelectrode is greater than a standard reduction potential of the secondcounter electrode.

In some embodiments, the sum of the area of the counter electrode andthe area of the second counter electrode is smaller than or equal to thearea of the working electrode.

In some embodiments, the counter electrode is a cathode, and thestandard reduction potential of an active material of the counterelectrode satisfies E_(s) ⁰>E_(m) ⁰−E_(v), where the E_(s) ⁰ is thestandard reduction potential of the active material, the E_(m) ⁰ is thestandard reduction potential for the concentration reaction on theworking electrode, and the E_(v) is the potential applied by a measuringapparatus when providing the measuring reaction.

In some embodiments, the counter electrode is an anode, and the standardreduction potential of an active material of the counter electrodesatisfies E_(s) ⁰<E_(m) ⁰−E_(v), wherein the E_(s) ⁰ is the standardreduction potential of the active material, the E_(m) ⁰ is the standardreduction potential for the concentration reaction on the workingelectrode, and the E_(v) is the potential applied by a measuringapparatus when providing the measuring reaction.

The present disclosure's biochemical test chip has a counter electrodewith a current density greater than the current density of the workingelectrode. Therefore, it is feasible to make the electrons oxidized orreduced by the counter electrode equal to the electrons reduced oroxidized by the working electrode without increasing the area of thecounter electrode. In this way, the present biochemical test chip canaddress the above-mentioned bottleneck effect and meet the current needfor reducing the sampling volume.

The present disclosure provides an electrochemical system, including anelectrode unit and a reactive unit electrically coupled to the electrodeunit. The electrode unit includes a working electrode and a counterelectrode, wherein a current density of the counter electrode is greaterthan a current density of the working electrode.

In some embodiments, the current density of the counter electrode isgreater than or equal to twice the current density of the workingelectrode.

In some embodiments, an area of the counter electrode is smaller than orequal to an area of the working electrode.

In some embodiments, the counter electrode is a cathode, and a standardreduction potential of an active material of the counter electrodesatisfies E_(s) ⁰>E_(m) ⁰−E_(v), where E_(s) ⁰ is a standard reductionpotential of the active material, E_(m) ⁰ is a standard reductionpotential for a concentration reaction on the working electrode, andE_(v) is a potential applied by a measuring apparatus when providing ameasuring reaction.

In some embodiments, the counter electrode is an anode, and a standardreduction potential of an active material of the counter electrodesatisfies E_(s) ⁰<E_(m) ⁰−E_(v), wherein E_(s) ⁰ is a standard reductionpotential of the active material, E_(m) ⁰ is a standard reductionpotential for a concentration reaction on the working electrode, andE_(v) is a potential applied by a measuring apparatus when providing ameasuring reaction.

In some embodiments, the reactive unit and a target analyte undergo aprimary reaction, and the counter electrode is configured to undergo asecondary reaction, wherein the secondary reaction does not interferewith the primary reaction, and the secondary reaction allows the counterelectrode to receive or release additional electrons.

In some embodiments, the electrochemical system further includes aprotective unit electrically coupled to the electrode unit, wherein theprotective unit is configured to oxidate the electrode unit after theelectrode unit receives an electron or to reduce the electrode unitafter the electrode unit loses an electron, wherein there is a potentialdifference (E_(cell) ⁰) between the protective unit and the electrodeunit.

In some embodiments, the potential difference (E_(cell) ⁰) is greaterthan 0.

The present disclosure provides an implantable biochemical test chip,including a substrate, a biocompatible coating, an electrode unit and areactive layer. The biocompatible coating is disposed over thesubstrate. The electrode unit is between the substrate and thebiocompatible coating, wherein the electrode unit comprises a workingelectrode and a counter electrode, wherein the counter electrode isconfigured to receive or release additional electrons through aself-secondary redox reaction, and a current density of the counterelectrode is greater than a current density of the working electrode.The reactive layer is electrically connected to the electrode unit.

In some embodiments, the implantable biochemical test chip furtherincludes a first end connected to a measuring apparatus and a second endimplanted into a specimen.

In some embodiments, the second end is covered by the biocompatiblecoating.

In some embodiments, the reactive layer is between the working electrodeand the counter electrode.

In some embodiments, the reactive layer covers the working electrode orthe counter electrode.

In some embodiments, the substrate has an elongated structure, and thebiocompatible coating surrounds the substrate.

In some embodiments, the electrode unit further comprises a spareelectrode over the substrate.

The present disclosure provides an implantable biochemical test chip,including a substrate, a biocompatible coating, an electrode unit and aprotective layer. The substrate has a measuring end and an implantableend. The biocompatible coating is disposed over the substrate. Theelectrode unit is between the substrate and the biocompatible coating,wherein the electrode unit comprises a working electrode and a counterelectrode, wherein the counter electrode includes an active material.The protective layer is electrically connected to the electrode unit,configured to stabilize the active material of the counter electrode.

In some embodiments, the protective layer is located at the measuringend.

In some embodiments, the protective layer is arranged adjacent to thecounter electrode.

In some embodiments, there is a potential difference (E_(cell) ⁰)between the protective layer and the electrode unit.

In some embodiments, the potential difference (E_(cell) ⁰) is greaterthan 0.

The foregoing outlines the technical features and advantages of thepresent disclosure so that those skilled in the art may betterunderstand the following detailed description of the presentapplication. Other technical features and advantages that constitute thesubject matter of the present disclosure are described below. Thoseskilled in the art should appreciate that they may readily use theconcepts and specific embodiments provided below as a basis fordesigning or modifying other structures and processes for carrying outthe same purposes and/or achieving the same advantages of the presentdisclosure. Those skilled in the art should also realize that suchequivalent constructions still fall within the spirit and scope of thepresent disclosure as defined in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the present disclosure are best understood from the followingdetailed description and claims when read with the accompanying figures.It is noted that, elements with the same reference numbers are the sameelements.

FIG. 1 is a schematic exploded view illustrating a biochemical test chipaccording to some embodiments of the present disclosure.

FIG. 2 is a partial top view illustrating a biochemical test chipaccording to some embodiments of the present disclosure.

FIG. 3A and FIG. 3B are schematic diagrams illustrating theelectrochemical reaction according to some embodiments of the presentdisclosure.

FIG. 4 is a partial top view illustrating a biochemical test chipaccording to some embodiments of the present disclosure.

FIG. 5 is a partial top view illustrating a biochemical test chipaccording to some embodiments of the present disclosure.

FIG. 6 is a partial top view illustrating a biochemical test chipaccording to some embodiments of the present disclosure

FIG. 7 is a schematic exploded view illustrating a biochemical test chipaccording to some embodiments of the present disclosure.

FIG. 8 is a partial top view illustrating a biochemical test chipaccording to some embodiments of the present disclosure.

FIGS. 9A and 9B are charts respectively showing the signals detected ona counter electrode according to the present embodiment and a counterelectrode of a comparative example under different concentrations.

FIG. 10 is a chart showing the signals detected on a counter electrodeaccording to the present embodiment and a counter electrode of acomparative example having different electrode areas.

FIG. 11A and FIG. 11B are charts respectively showing the signalsdetected on an anode counter electrode and a cathode counter electrodewith different materials.

FIG. 12 is a schematic diagram illustrating an electrochemical systemaccording to some embodiments of the present disclosure.

FIG. 13 is a schematic view illustrating an implantable biochemical testchip according to some embodiments of the present disclosure.

FIG. 14A is a cross-sectional view taken along line A-A′ of FIG. 13according to some embodiments of the present disclosure.

FIG. 14B is a cross-sectional view taken along line B-B′ of FIG. 13according to some embodiments of the present disclosure.

FIG. 15 is a schematic diagram illustrating an electrochemical reactionof the electrochemical system or the implantable biochemical test chipaccording to some embodiments of the present disclosure.

FIG. 16 is a schematic view illustrating an implantable biochemical testchip according to some embodiments of the present disclosure.

FIG. 17 is a cross-sectional view taken along line C-C′ of FIG. 16according to some embodiments of the present disclosure.

FIG. 18 is a schematic view illustrating an implantable biochemical testchip according to some embodiments of the present disclosure.

FIG. 19 is a schematic exploded view illustrating an implantablebiochemical test chip according to some embodiments of the presentdisclosure.

FIG. 20 is a cross-sectional view taken along line D-D′ of FIG. 19according to some embodiments of the present disclosure.

FIG. 21 is a schematic exploded view illustrating an implantablebiochemical test chip according to some embodiments of the presentdisclosure.

FIG. 22 is a schematic view illustrating an implantable biochemical testchip according to some embodiments of the present disclosure.

FIG. 23 is a schematic view illustrating an implantable biochemical testchip according to some embodiments of the present disclosure.

FIG. 24 is a schematic exploded view illustrating an implantablebiochemical test chip according to some embodiments of the presentdisclosure.

FIG. 25 is a chart showing signals detected on a counter electrodeaccording to the present embodiment and a counter electrode of acomparative example under different concentrations.

FIG. 26 is a chart showing signals detected on an electrochemical systemaccording to the present embodiment and an electrochemical system of acomparative example over time.

DETAILED DESCRIPTION

Detailed description of the present disclosure is discussed in detailbelow. However, it should be understood that the embodiments providemany inventive concepts that can be applied in a variety of specificcontexts. The specific embodiments discussed are illustrative of thespecific ways they can be made and used and do not limit the presentdisclosure's scope.

The same reference numeral is configured to represent the sameelements/components in the various drawings and illustrativeembodiments. Reference will now be made in detail to the illustrativeembodiments shown in the drawings. Whenever possible, the same referencenumeral is used in the drawings and the specification to represent thesame or similar parts. In the drawings, the shape and thickness may beexaggerated for clarity and convenience. The description will bedirected specifically to the elements forming part of, or more directlycooperating with, the device disclosed hereunder. As could beappreciated, elements not explicitly shown or described may take variousforms. The reference to “some embodiments” or “embodiment” throughoutthis specification implies that the particular features, structures, orcharacteristics described in conjunction with the embodiment areincluded in at least one of the embodiments. Therefore, the phrase “insome embodiments” or “in an embodiment” appearing in various placesthroughout this specification does not necessarily refer to the sameembodiment. Besides, the specific features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

In the drawings, the same reference numeral is configured to indicatethe same or similar elements in the various views, and illustrativeembodiments of the present application are shown and described. Thedrawings are not necessarily drawn to scale, and in some cases, thedrawings have been exaggerated and/or simplified and are configured forillustrative purposes only. Many possible applications and variations ofthe present application will be understood by those of ordinary skill inthe art in view of the following illustrative embodiments of the presentdisclosure.

Unless otherwise defined, all terms used herein, including technical andscientific terms, have the same meanings as those commonly understood bya person of ordinary skill in the art in the field of the disclosedembodiments. It should be understood, for example, that terms defined incommon dictionaries should be construed to have meanings consistent withtheir meanings in the relevant field and context of this disclosure andshould not be construed or understood to have meanings that are tooformal unless expressly defined herein.

Besides, the following embodiments are provided to illustrate the corevalue of this disclosure but are not intended to limit the scope ofprotection of this disclosure. For clarity and ease of understanding,the same or similar functions or elements among this disclosure'sdifferent embodiments are not repeated or shown in the drawings.Besides, different elements or technical features from differentembodiments may be combined or substituted to create further embodimentsthat are still covered by this disclosure, provided they do not conflictwith each other.

The present disclosure is directed to an electrochemical system in whichthe counter electrode undergoes the self-secondary redox reaction toprovide additional electrons; in particular, to a biochemical test chiputilizing the electrochemical system in which the counter electrodeundergoes the self-secondary redox reaction. Furthermore, the presentdisclosure is directed to a counter electrode having an active material,which is capable of providing the same amount of electrons generated bythe reaction of the conductive medium on the working electrode by itsown secondary redox reaction when the electrode area is limited or whenthe concentration of the conductive medium on the surface of the counterelectrode in the reaction solution is insufficient for electrontransfer. As a result, the counter electrode's capability in balancingthe electric neutrality can be improved, and the electrochemical circuitcan be stabilized to avoid a current bottleneck effect on the counterelectrode. In some embodiments, the biochemical test chip furtherincludes a protective layer to help stabilize the active material on thecounter electrode, thereby protecting the biochemical test chip andmitigating or avoiding unexpected alterations in the biochemical testchip and the environment.

Reference is made to FIG. 1 ; FIG. 1 is a schematic exploded viewillustrating a biochemical test chip 100 according to some embodimentsof the present disclosure. The biochemical test chip 100 can be anelectrochemical test chip, which is a device that can be electricallyconnected to. The biochemical test chip 100 is configured to collect aspecimen and carry out electrochemical reaction therewith so as todetect a target analyte therein. The specimen includes any liquids orsoluble solids having therein one target analyte that can be detectedusing an electrochemical method. For example, the specimen may includeblood, tissue fluid, urine, sweat, tears, and other biological samples;however, the present disclosure is not limited thereto. Moreover, theblood can include the whole blood, plasma, serum, etc.; however, thepresent disclosure is not limited thereto.

Reference is made to FIG. 1 ; the biochemical test chip 100 includes aninsulating substrate 10, an electrode unit 20, a first insulating septum30, a reactive layer 40, and a second insulating septum 50. Theinsulating substrate 10 includes a substrate that is electricallyinsulated. In some embodiments, the material of the insulating substrate10 can include polyvinyl chloride (PVC), glass fiber (FR-4),polyethersulfone (PES), bakelite, polyethylene terephthalate (PET),polycarbonate (PC), polypropylene (PP), polyethylene (PE), polystyrene(PS), polyimide (PI), glass plate, ceramic or any combination of theabove-mentioned materials; however, the present disclosure is notlimited thereto. The material of the insulating substrate 10 can beadjusted depending on the system or actual needs.

The electrode unit 20 of the biochemical test chip 100 is located on theinsulating substrate 10. The electrode unit 20 is disposed on theinsulating substrate 10 and configured to be subjected to theelectrochemical measurement. The electrochemical measurement includesanalyzing the specimen's concentration using an electrical reaction,such as potentiometry, conductometry, voltammetry, polarimetry,high-frequency titration, amperometry, Coulombic method, electrolysisand the like. The electrode unit 20 includes a working electrode 22 anda counter electrode 24; however, the present disclosure is not limitedthereto. The electrode unit 20 can have other electrodes depending onthe requirements of the system. The working electrode 22 is theelectrode that allows the target analyte to undergo theelectro-oxidation reaction or electro-reduction reaction on the surfacethereof and can be used by the measuring apparatus to determine theconcentration. In detail, the electro-oxidation reaction orelectro-reduction reaction is an electrochemical reaction in which thetarget analyte undergoes an exchange between electrical and chemicalenergy on the surface of the working electrode 22.

The polarity of the working electrode 22 can be an anode or a cathode,depending on the requirement of the measurement reaction. For example,if the target analyte is oxidized on the working electrode 22, theworking electrode 22 is an anode; if the target analyte is reduced onthe working electrode 22, the working electrode 22 is a cathode. Thecounter electrode 24 is an electrode that undergoes theelectro-reduction reaction or electro-oxidation reaction correspondingto the working electrode 22 so that the overall electrochemical systemsatisfies the principles of charge balance. The potential and polarityof the counter electrode 24 are opposite to the potential and thepolarity of the working electrode 22. Before being in contact with thespecimen, the working electrode 22 and the counter electrode 24 areinsulated from each other. After the working electrode 22 and thecounter electrode 24 are in in contact with the specimen, they form anelectrical loop with the measuring apparatus. In some embodiments, theworking electrode 22 and the counter electrode 24 can include a carbonelectrode, silver electrode, platinum electrode, etc.; however, thepresent disclosure is not limited thereto. The materials of the workingelectrode 22 and the counter electrode 24 can vary depending on thesystem's requirement.

The first insulating septum 30 is disposed on the insulating substrate10 and located on the electrode unit 20. The first insulating septum 30can have an opening 32, wherein the opening 32 at least partiallyexposes the electrode unit 20. In some embodiments, the opening 32 islocated the front side 30F of the first insulating septum 30 and exposesa portion of the electrode unit 20. The opening 32 is configured todefine a reaction zone 34 in the biochemical test chip 100; the reactionzone 34 is configured to accommodate the specimen. The portion of theelectrode unit 20 exposed from the opening 32 can undergo theelectrochemical reaction with the specimen. The size of shape of theopening 32 can be adjusted according to the desired area of theelectrode unit 20 and the desired volume of the specimen. In someembodiments, the back side 30B of the first insulating septum 30 exposesa portion of the electrode unit 20 to form a connecting zone 10C. Theelectrode unit 20 exposed from the connecting zone 10C can beelectrically connected to the measuring apparatus. The measuringapparatus and the biochemical test chip 100 are electrically connectedto provide the energy required for the electrochemical measurement andanalyze the reaction signal. In some embodiments, the material of thefirst insulating septum 30 includes a PVC insulation tape, PETinsulation tape, heat drying insulation paint or ultraviolet (UV)curable insulation paint; however, the present disclosure is not limitedthereto.

FIG. 2 is a partial top view illustrating the biochemical test chip 100according to some embodiments of the present disclosure. Reference ismade to FIG. 2 and FIG. 1 simultaneously; the biochemical test chip 100further includes a reactive layer 40. The reactive layer 40 is locatedin the opening 32 of the first insulating septum 30. The reactive layer40 is located in the reaction zone 34. The reactive layer 40 isconfigured to undergo a chemical reaction with the specimen. Thereactive layer 40 is electrically connected to the electrode unit 20. Insome embodiments, the reactive layer 40 is electrically connected to theworking electrode 22 of the electrode unit 20. In some embodiments, thearea of the reactive layer 40 is smaller than the size of the opening32. The reactive layer 40 at least partially covers the electrode unit20 exposed from the opening 32. In the present embodiment, the reactivelayer 40 only covers the working electrode 22; however, the presentdisclosure is not limited thereto. In some embodiments, the reactivelayer 40 at least partially contacts the working electrode 22 of theelectrode unit 20. In some embodiments, the reactive layer 40 at leastpartially contacts the working electrode 22 and the counter electrode 24of the electrode unit 20.

In some embodiments, the reactive layer 40 includes an enzyme and aconductive medium. For example, the enzyme includes a fixed or non-fixedenzyme, such as redox enzymes, antigens, antibodies, microbial cells,animal and plant cells, and biologically identifiable components ofanimal and plant tissues. The conductive medium is configured to receiveelectron generated after the reaction between the enzyme and a bloodspecimen and transmit the electrons to the measuring apparatus via theelectrode unit 20. In some embodiments, the conductive medium caninclude potassium hexacyanoferrate(III), potassium hexacyanoferrate(II)trihydrate, ruthenium complex, ferrocene, sodium dithionite,nicotinamide adenine dinucleotide (NAD+), nicotinamide adeninedinucleotide phosphate (NADP+), thiamin pyrophosphate (TPP), coenzyme A(HSCoA), flavin adenine dinucleotide (FAD) or a combination thereof,however, the present disclosure is not limited thereto. In someembodiments, the reactive layer 40 can be further supplemented with aphosphate buffer and protectants, such as, protein, dextrin, glucan,amino acid, etc.; however, the present disclosure is not limitedthereto.

Reference is made again to FIG. 1 , the second insulating septum 50 islocated on the first insulating septum 30. In some embodiments, thesecond insulating septum 50 at least partially covers the opening 32 ofthe first insulating septum 30, so that the opening 32 forms a capillarystructure. In some embodiments, the terminus of the second insulatingseptum 50 is disposed with a vent 502 corresponding to the opening 32.The vent 52 can be of any shapes; for example, the vent 52 can becircular, oval, rectangular, rhombus, etc. The second insulating septum50 can be of any shapes or sizes. In some embodiments, the secondinsulating septum 50 also exposes the connecting zone 10C of theelectrode unit 20.

Reference is made to FIG. 2 and FIG. 1 simultaneously; the opening 32 ofthe first insulating septum 30 at least partially exposes the electrodeunit 20. The opening 32 at least partially exposes the working electrode22 and the counter electrode 24. In the present embodiment, at least theworking electrode 22 and the counter electrode 24 are disposed in thereaction zone 34; however, the present disclosure is not limitedthereto. In other embodiments, electrodes having other functions can befurther disposed in the reaction zone 34. Moreover, the presentdisclosure does not particularly limit the configuration of theelectrodes; the working electrode 22 and the counter electrode 24 can beof any shapes. In some embodiments, the working electrode 22 and thecounter electrode 24 can have different shapes. In some embodiments, thematerial of the working electrode 22 is different from or the same asthe material of the counter electrode 24.

In the present embodiment, the working electrode 22 and the counterelectrode 24 are insulated from each other before being in contact withthe specimen. When the specimen contacts the working electrode 22 andthe counter electrode 24, the working electrode 22 and the counterelectrode 24 form an electrical loop with the measuring apparatus. FIG.3A and FIG. 3B are schematic diagrams illustrating the electrochemicalreaction according to some embodiments of the present disclosure. Forsimplification, FIG. 3A and FIG. 3B only illustrate a portion of thebiochemical test chip 100. In detail, FIG. 3A and FIG. 3B onlyillustrates the portions of the working electrode 22 and the counterelectrode 24 within the reaction zone 34. As shown in FIG. 3A and FIG.3B, the working electrode 22 and the counter electrode 24 exposed fromthe reaction zone 34 are in in contact with the specimen S and form anelectrical loop with the measuring apparatus M. FIG. 3A and FIG. 3Bfurther illustrate the portions of the enzyme E and the conductivemedium C within the reaction zone 34, wherein the enzyme E and theconductive medium C form a portion of the reactive layer 40 (shown inFIG. 2 ). In some embodiments, the conductive medium C can be ironirons; however, the present disclosure is not limited thereto.

Reference is made to FIG. 3A, the specimen S includes a target analyteR, wherein the target analyte R includes an electrochemically activesubstance or electrochemically reactive substance. After the specimen Sis loaded into the reaction zone 34 of the biochemical test chip 100,the target analyte R in the specimen S is reduced by the enzyme E in thereactive layer 40, thereby forming a reduced target analyte R′; however,the present disclosure is not limited thereto. In other embodiments, thetarget analyte R in the specimen S can be oxidized by the enzyme E inthe reactive layer 40, thereby forming an oxidized target analyte R″.

The conductive medium C of the reactive layer 40 is configured toreceive or provide the electrons generat4ed or lost due to the reactionbetween the enzyme E and the target analyte R in the specimen S andtransmit the electrons to the measuring apparatus M via the workingelectrode 22 of the electrode unit 20. For simplification, theconductive medium C in this embodiment include, for example, bivalentferrous irons (Fe²⁺); however, the present disclosure is not limitedthereto. When the target analyte R in the specimen S is reduced, theconductive medium C of the reactive layer 40 is oxidized. In the presentembodiment, bivalent ferrous irons (Fe²⁺) are oxidized into trivalentferric irons (Fe³⁺). Moreover, the conductive medium C of the reactivelayer 40 is oxidized in an amount corresponding to the amount in whichthe target analyte R is reduced.

When the conductive medium C of the reactive layer 40 (e.g., Fe²⁺)releases electrons into an oxidized conductive medium C′ (e.g., Fe³⁺),the working electrode 22 also undergoes a reaction to reduce theoxidized conductive medium C′ (e.g., Fe³⁺) into the reduced conductivemedium C (e.g., Fe²⁺). The measuring apparatus M detects the changes inthe number of electrons (e⁻) generated by the reaction on the workingelectrode 22 and carries out the concentration analysis. When theworking electrode 22 undergoes the reduction reaction, the counterelectrode 24 must oxidize a corresponding amount of the reducedconductive medium C (e.g., Fe²⁺), so that the overall reaction achievesthe electrical neutral balance.

Generally, when the oxidizabilty of the counter electrode 24 isinsufficient to match the working electrode 22; e.g., the amount of theconductive medium C oxidized by the counter electrode 24 is smaller thanthe amount of the conductive medium C reduced by the working electrode22, the level of the reducing current generated by the working electrode22 is limited due to the principle of electrical neutrality, therebysuffering from the bottle-neck effect. The bottleneck effect caused bythe working electrode 22 reacting with more electron flow than thecounter electrode 24 can react with will limit the range of themeasurable concentration of the biochemical test chip 100.

In the prior art, the occurrence of the aforementioned bottleneck effectis mostly related to the size of the working electrode 22 and thecounter electrode 24. The electrochemical oxidation or reductionreaction of the target analyte R in the specimen S generates a certaincurrent change, which is linearly proportional to the concentration ofthe target analyte R in the specimen S. Therefore, the concentration ofthe target analyte R in the specimen S can be analyzed by measuring theoxidation or reduction current on the surface of the working electrode22, and the relationship between current and concentration can beexpressed as:

${i = \frac{{nFAC}_{0}\sqrt{D}}{\sqrt{\pi t}}},$

where, I is the measured current (unit: A); n is the number of theelectrons generated during the redox reaction; F is the Faraday constant(96500 C/mol); A is the surface area of the working electrode (unit:cm²); C₀ is the initial concentration of the specimen (unit: mol/cm³); Dis the diffusion constant (unit: cm²/s); t is time (unit: s). in view ofthe foregoing, the level of the current signal of the electrochemicalreaction is proportional to the surface area of the electrode.

In the prior art, in order to save the manufacturing process, theworking electrode 22 and the counter electrode 24 are mostly made of thesame material. In addition, to avoid additional background signalinterference caused by oxidation reduction of the electrode materialitself and to consider the lifetime of the electrode, the material ofthe working electrode 22 and the counter electrode 24 are mostly made ofinert carbon. In other embodiments, the materials of the workingelectrodes 22 and counter electrodes 24 include gold, palladium, etc. Inother prior art embodiments, the material of the working electrode 22 ismostly selected from materials that are more reactive with the targetanalyte R of the specimen S compared to the material of the counterelectrode 24. The material of the working electrode 22 is often selectedto have a better electrochemical activity or electrochemical reactivitythan the material of the counter electrode 24. For example, the materialof the working electrode 22 is mostly a silver electrode or silver oxideelectrode that has better reactivity with the target analyte R, whilethe material of the counter electrode 24 is mostly a carbon electrode orplatinum electrode that has low reactivity with the target analyte R.

Reference is made to FIG. 3B, in the present disclosure, the material ofthe working electrode 22 and the counter electrode 24 is chosen so thatthe current density of the counter electrode 24 is greater than thecurrent density of the working electrode 22. In some embodiments, thematerial of the counter electrode 24 has better electrochemicalreactivity compared to that of the working electrode 22. In detail, thematerial of the counter electrode 24 is selected to have a betterelectrochemical reactivity with environmental substances. Saidenvironmental substances refer to substances that are not the targetanalyte R of the specimen S. In some embodiments, the material of thecounter electrode 24 has a better electrochemical activity than thematerial of the working electrode 22. The material of the counterelectrode 24 is selected to have a better electrochemical reactivitywith the material of the environmental substance. In some embodiments,the area of the counter electrode 24 may be smaller than or equal to thearea of the working electrode 22. In some embodiments, the currentdensity of the counter electrode 24 is greater than or equal to twotimes the current density of the working electrode 22.

In the present embodiment, the counter electrode 24 can include aconductive active material. In some embodiments, the active material maybe doped in the counter electrode 24. In some embodiments, the activematerial may be formed on the surface of the counter electrode 24. Insome embodiments, the counter electrode 24 is composed of the activematerial. The purpose of using the active material is that when thebiochemical test chip 100 undergoes an electrochemical reaction, theactive material of the counter electrode 24 can carry out its ownoxidation or reduction reaction without interfering with the primaryreaction. Said primary reaction refers to the oxidation or reductionreaction caused by the target analyte R and the reaction layer 40, andsaid secondary reaction refers to the oxidation or reduction reactionthat is not caused by the target analyte R and the reaction layer 40. Indetail, as long as the secondary reaction occurring on the counterelectrode 24 does not affect the primary reaction of the workingelectrode 22, the source of the reactants is not limited. Therefore, thematerials required for the secondary reaction can come from either thespecimen S or the environment. The active material of the counterelectrode 24 refers to a substance that can undergo redox within theworking voltage range. The working voltage refers to the voltageprovided by the measuring apparatus M to cause the working electrode 22and the counter electrode 24 to perform the electrochemical reaction. Insome embodiments, the working voltage is ±10 volts (V). In otherembodiments, the working voltage is ±5V. In some embodiments, theworking voltage is ±2V. In some embodiments, the working voltage is ±1V.

Reference is made to FIG. 3B. Since the counter electrode 24 of thepresent embodiment includes a conductive active material, it can andundergo the secondary reaction without interfering the primary reaction.Said secondary reaction allows the counter electrode 24 to have theability to receive or release additional electrons. Said additionalelectrons refer to electrons not generated by the enzyme E or theconductive medium C of the reactive layer 40. In other words, inaddition to undergo the electrochemical reaction with the target analyteR in the specimen S, the counter electrode 24 can obtain electrons byundergoing the secondary reaction with the active material withoutdisrupting the electrical neutrality of the measuring apparatus M.

As shown in FIG. 3B, after the specimen S is filled in the reaction zone34 and the measuring apparatus M supplies the working voltage, thetarget analyte R in the specimen S and the enzyme E of the reactivelayer 40 undergo the reaction, and a corresponding amount of theoxidized conductive medium C′ on the working electrode 22 is reducedinto the reduced conductive medium C. On the other hand, the counterelectrode 24 needs to oxidize the reduced conductive medium C to theoxidized conductive medium C′ in an amount that is corresponding to thenumber of reduced electrons of the working electrode 22 to maintain theelectrical neutrality of the overall system. Since the counter electrode24 of the present disclosure has an active material that can undergo thesecondary reaction, when the counter electrode 24 oxidizes the reducedconductive medium C and receives electrons, it also performs anoxidation reaction at the same time to generate additional electrons. Inthis embodiment, the materials of the working electrode 22 and thecounter electrode 24 are selected so that the current density of thecounter electrode 24 is greater than the current density of the workingelectrode 22. The active material of the counter electrode 24 canperform its own oxidation or reduction reaction without interfering withthe primary reaction.

Reference is made again to FIG. 1 and FIG. 2 , the working electrode 22is an electrode for analyzing electro-oxidation or electro-reductioncurrent. Therefore, the working electrode 22 is composed of an inactivematerial that does not cause interference on the electrode surface. Itis worth noting that the inactive material referred to in the presentdisclosure means that the material will not undergo oxidation orreduction reactions in the measurement environment of the biochemicaltest chip 100. In other words, the inactive material referred to in thisdisclosure may have redox capability in other specific environments, butthe inactive material will not undergo redox reaction during themeasurement process according to the present disclosure. The inactivematerial of the working electrode 22 may include a conductive material.For example, the inactive material of the working electrode 22 mayinclude palladium, platinum, gold, carbon or a combination thereof, butthe disclosure is not limited thereto. The inactive material of theworking electrode 22 can be adjusted depending on system requirements.

On the other hand, the active material of the counter electrode 24refers to the material that undergoes oxidation or reduction in themeasurement environment of the biochemical test chip 100. The redoxchange of the active material of the counter electrode 24 has a director indirect causal relationship with the measurement environment of thebiochemical test chip 100. In the present disclosure, thecharacteristics of the material of the counter electrode 24 must matchthe characteristics of the working electrode 22. For example, if theworking electrode 22 performs an electro-reduction reaction, the activematerial of the counter electrode 24 must be a material with its ownoxidizing ability. In other embodiments, if the surface of the workingelectrode 22 undergoes an electro-oxidation reaction, the counterelectrode 24 must be a material with its own reducing ability.

It is worth noting that, in order to ensure that the active material ofthe counter electrode 24 can perform the preset function during theelectrochemical measurement, excessive reaction before the measurementshould be avoided; so, the active material of the counter electrode 24of the present disclosure is in contact with the reaction layer 40 toprevent the two from reacting with each other before measurement. Toprevent the two from reacting before the measurement. However, duringthe electrochemical measurement of the biochemical test chip 100, thereaction layer 40 also needs to react with the counter electrode 24, sothe smaller the distance between the working electrode 22 and thecounter electrode 24, the better. In some embodiments, the activematerial of the counter electrode 24 may be covered with a protectivefilm, wherein the protective film may melt after the specimen S enters.

In some embodiments, the active material of the counter electrode 24 caninclude, but is not limited to, silver (Ag), tin (Sn), iron (Fe), zinc(Zn), cobalt (Co), nickel (Ni), lead (Pb), copper (Cu), manganesedioxide (MnO₂), ferroferric oxide (Fe₃O₄), ferric oxide (Fe₂O₃), ferrousoxide (FeO), silver chloride (AgCl), cobalt trioxide (CO₂O₃), cobalt(II) oxide (CoO), nickle (III) oxide (Ni₂O₃), nickle (II) oxide (NiO),copper oxide (CuO), cuprous oxide (Cu₂₀), benzoquinone, ferrocene,ferrocenium, spinel structure mix-valence metal oxides (e.g., Fe₃O₄,CO₃O₄, etc.), ferrocyanide, Prussian blue (Fe₄[Fe(CN)₆]₃), metalferricyanides ([Fe(CN)₆]³⁻), metal ferrocyanides ([Fe(CN)₆]⁴⁻), metalcomplexes, or a combination thereof, however, the present disclosure isnot limited thereto.

Reference is made again to FIG. 3B; for simplification, silver (Ag) isused as an example of the active material of the counter electrode 24;however, the present disclosure is not limited thereto. In detail, sincesilver (Ag) in the counter electrode 24 is in direct contact with thespecimen S, in addition to the working voltage applied by the measuringapparatus M, the reaction zone 34 further comprises water (H₂O) orhydroxide ions (OH⁻) that allow silver (Ag) to be oxidized. Therefore,under appropriate conditions, silver in the counter electrode 24 canundergo oxidation reaction with the hydroxide ions (OH⁻), water (H₂O) orwater vapor from the specimen S or the environment, and the reactionformula can be expressed as 2Ag+2OH⁻ →Ag₂O+H₂O+2e⁻ or2Ag+H₂O→Ag₂O+2H⁺+2e⁻. Silver oxide (Ag₂O), water (H₂O) and electrons aregenerated after the silver and hydroxide ions (OH⁻) or water from thespecimen S or the environment undergo redox reaction. Therefore, withrespect to the overall reaction, the counter electrode 22 is alsoself-oxidized to form the oxidized electrode (silver oxide) and releaseelectrons (e⁻), in addition to oxidize the reduced conductive medium C(e.g., Fe²⁺). Hence, electrons generated by the active material of thecounter electrode 24 help to improve the overall electrical neutralityof the counter electrode 24 under the principle of conservation ofcharge.

Thus, when the working electrode 22 undergoes extensiveelectro-reduction, the counter electrode 24 can undergoelectro-oxidation correspondingly to satisfy the overall system'selectrical neutrality. It should be noted that although the activematerial (e.g., silver) of the counter electrode 24 consumes water (H₂O)or hydroxide ions (OH⁻) and produces hydrogen ions (H⁺) or water (H₂O)during the oxidation process, and thereby partially changes the pH ofthe reaction zone 34, the resulted change in pH is minimal for theoverall system. Hence, the pH change does not affect the primaryreaction and the test results, and can be ignored.

To ensure that the active material of the counter electrode 24 has theabove-mentioned capabilities, the active material and the counterelectrode 24 must have the same reaction polarity. In other words,although the change in the oxidation number of the active material andthe counter electrode 24 may not be equal, when the counter electrode 24is an anode, the active material must have oxidizing ability; when thecounter electrode 24 is a cathode, the active material must havereducing ability. Therefore, the selection of the active material needsto match the reaction polarity of the counter electrode 24, and it mustbe chosen to improve the electrical neutrality ability of the counterelectrode 24 under the principle of conservation of charge. Therefore,when the counter electrode 24 is an anode, the standard reductionpotential of the active material of the counter electrode 24 mustsatisfy E_(s) ⁰>E_(m) ⁰−E_(v). Here, E_(s) ⁰ is the standard reductionpotential of the active material, E_(m) ⁰ is the standard reductionpotential of the concentration reaction on the working electrode 22;E_(v) is the potential applied by the measuring apparatus M whenproviding the measuring reaction.

In some embodiments, the conductive medium C is ferrocyanide(Fe^(II)(CN)₆ ⁴⁻), the working voltage (E_(v)) applied by the measuringapparatus M is +0.4V, the working electrode 22 undergoes oxidationreaction, and hence the reaction of the conductive medium C on thesurface of the working electrode 22 is Fe^(II)(CN)₆ ⁴⁻→Fe^(III)(CN)₆³⁻+e⁻, wherein E_(m) ⁰=0.36V. Therefore, in the present embodiment, thestandard reduction potential (E_(s) ⁰) of the active material of thecounter electrode 24 undergoing the reduction reaction must be greaterthan −0.04V; thus, the material with a standard reduction potential(E_(s) ⁰) greater than −0.04V can be chosen as the material for thecounter electrode 24, such as, Fe₃O₄ (E⁰=+0.085V), AgCl (E⁰=+0.2223V),ferrocenium (E⁰=+0.4V), benzoquinone (E⁰=+0.6992V) and the like;however, the present disclosure is not limited thereto.

In some embodiments, the conductive medium C is ferricyanide(Fe^(III)(CN)₆ ³⁻), the working voltage (E_(v)) applied by the measuringapparatus M is −0.4V, the working electrode 22 undergoes reductionreaction, and hence the reaction of the conductive medium C on thesurface of the working electrode 22 is Fe^(III)(CN)₆ ³⁻+e⁻→Fe^(II)(CN)₆⁴⁻, wherein E_(m) ⁰=0.36 V. Therefore, in the present embodiment, thestandard reduction potential (E_(s) ⁰) of the active material of thecounter electrode 24 undergoing the reduction reaction must be less than0.76V; thus, the material with a standard reduction potential (E_(s) ⁰)smaller than 0.76V can be chosen as the material for the counterelectrode 24, such as, ferrocene (E⁰=+0.4V), Cu (E⁰=+0.34V), Fe(E⁰=+0.085V), Sn (E⁰=−0.1V), and the like; however, the presentdisclosure is not limited thereto.

The foregoing is only an example of the active material of the counterelectrode 24, and the present disclosure is not limited thereto.Moreover, the standard reduction potential of the active material of thecounter electrode 24 is not limited to the foregoing. As discussedabove, one should consider the polarity of the active material of thecounter electrode 24, wherein the polarity of active material should bethe same as the polarity of the measuring reaction on the counterelectrode 24. Further, the standard reduction potential of the activematerial of the counter electrode 24 should satisfy the condition ofE_(s) ⁰<E_(m) ⁰−E_(v) or E_(s) ⁰>E_(m) ⁰−E_(v). In some embodiments,E_(v) can be ±5 V˜±2 mV. In some embodiments, E_(v) can be ±2 V˜±80 mV.In some embodiments, E_(v) can be ±0.8 V˜±0.1 V.

The present disclosure is not limited to the foregoing embodiments, andcan comprise other different embodiments. For simplification purposesand to facilitate the comparison among embodiments of the presentdisclosure, in the following embodiments, each of the completely thesame elements is labeled with completely the same reference numeral. Tofurther facilitate the comparison among the differences between theseembodiments, only the differences among different embodiments arediscussed, whereas the completely the same features are not discussedfor the sake of brevity.

FIG. 4 is a partial top view illustrating a biochemical test chipaccording to some embodiments of the present disclosure. As shown inFIG. 4 , the difference between the biochemical test chip 200 and thebiochemical test chip 100 is that the counter electrode 24 includes afirst portion 24A and a second portion 24B. In some embodiments, thecounter electrode 24 can include inactive material and active material.For example, in the present embodiment, the first portion 24A of thecounter electrode 24 includes an active material, whereas the secondportion 24B includes an inactive material. Therefore, the first portion24A is capable of receiving or releasing additional electrons.

In some embodiments, the active material can be disposed on theinsulating substrate 10 first, and then the inactive material can beapplied onto a predetermined position to form the first portion 24A andthe second portion 24B of the counter electrode 24 according to thepresent embodiment. In some embodiments, the inactive material can bedisposed on the insulating substrate 10 first, and then the activematerial are disposed on the predetermined location of the opening 32 toform the first portion 24A and the second portion 24B of the counterelectrode 24 according to the present embodiment. In this way, the firstportion 24A (active material) of the counter electrode 24 is exposedfrom the opening 32.

The method for disposing the first portion 24A and the second portion24B of the counter electrode 24 can include techniques like screenprinting, imprinting, thermal transfer printing, spin coating, ink-jetprinting, laser ablation, deposition, electrodeposition, etc.; however,the present disclosure is not limited thereto. In some embodiments,monomers with redox capability are polymerized on the inactive materialsurface of the counter electrode 24 using plasma or other means forchemical bonding modification to form polymers, such as polyaniline,polypyrrole, polythiophene, polyvinylferrocene, etc.; however, thepresent disclosure is not limited thereto. In some embodiments, theinactive material surface of the counter electrode 24 can be graftedwith high molecular chains and bonded with conductive medium with redoxcapabilities (such as, ferrocenecarboxylic acid, to form the counterelectrode 24.

FIG. 5 is a partial top view illustrating a biochemical test chipaccording to some embodiments of the present disclosure. As shown inFIG. 5 , the difference between the biochemical test chip 300 and thebiochemical test chip 200 is that the biochemical test chip 100 furtherincluding the protective layer 60. For example, the counter electrode 24exposed to the environment may get spoiled upon being oxidized with thewater vapor or oxygen in the air. The biochemical test chip 300 includesan additional protective layer 60 to protect the stability of the activematerial of the counter electrode 24. The protective layer 60 can beconfigured to protect the counter electrode 24 in the biochemical testchip 300 to slow the unexpected spoilage of the first portion 24A of thecounter electrode 24 in the environment, wherein said spoilage mayresult in no or reduced receipt or release of additional electrons.

The protective layer 60 is disposed on specific regions in the electrodeunit 20. In some embodiments, the protective layer 60 electricallyconnected to the first portion 24A of the counter electrode 24 via theelectrode unit 20. For example, in the present embodiment, theprotective layer 60 is electrically connected to the first portion 24Aand the second portion 24B of the counter electrode 24 via the branch24C of the counter electrode 24. In some embodiments, the counterelectrode 24 do not have the branch 24C, and the protective layer 60 canbe disposed directly on the second portion 24B of the counter electrode24 so that it is electrically connected to the first portion 24A of thecounter electrode 24. In some embodiments, the protective layer 60 andthe first portion 24A of the counter electrode 24 locate on the samelevel. In some embodiments, the protective layer 60 and the firstportion 24A of the counter electrode 24 locate on different levels. Forexample, the protective layer 60 can be disposed above or under thecounter electrode 24. In some embodiments, the protective layer 60 canbe surrounded by the second portion 24B of the counter electrode 24.

In some embodiments, the first insulating septum 30 can have a firstopening (not shown in the drawings), and the second insulating septum 50can have a second opening (not shown in the drawings), wherein the firstopening and second opening at least partially expose the protectivelayer 60. The protective layer 60 and the counter electrode 24 can beexposed to the same environment; however, the present disclosure is notlimited thereto. For example, the protective layer 60 can be disposedbetween the insulating substrate 10 and the first insulating septum 30,and is exposed to same environment as the first portion 24A of thecounter electrode 24 via the first opening and the second opening. Inother embodiments, the protective layer 60 and the counter electrode 24can be exposed to different environment. For example, the protectivelayer 60 can be disposed between the insulating substrate 10 and thefirst insulating septum 30, and the first insulating septum 30 and thesecond insulating septum 50 do not have the first opening and the secondopening. The position of the protective layer 60 is not limited to thosedescribed the above, in some embodiments, the protective layer 60 can bedisposed on the second insulating septum 50 and electrically connectedto the electrode unit 20 via wires. In other embodiments, the protectivelayer 60 can be disposed between the first insulating septum 30 and thesecond insulating septum 50 and electrically connected to the electrodeunit 20 via wires.

The protective layer 60 can take the forms of solid, liquid, or gas. Forexample, solids can include pure metals, alloys, metal compounds(halides, oxidates, mixed-valence compounds, organometallic complexes),organic redox agents, etc. Liquids can include aqueous solutions,organic solutions, supercritical fluids, liquid elements (e.g., bromine,mercury), etc. Gases can include gaseous elements (e.g., oxygen, ozone),gaseous compounds (e.g., ammonium, nitrogen monoxides) etc.

The protective layer 60 and the counter electrode 24 (or the firstportion 24A of the counter electrode 24) may have different materials orcompositions. There can be a potential difference (E_(cell) ⁰) betweenthe protective layer 60 and the counter electrode 24. In someembodiments, there is a potential difference (E_(cell) ⁰) between theprotective layer 60 and the first portion 24A of the counter electrode24. The potential difference (E_(cell) ⁰) can be expressed as E_(cell)⁰=E_(cathode)−E_(anode), where E_(cathode) is the standard reductionpotential of the cathode (the cathode electrode), and E_(anode) is thestandard reduction potential of the anode (the anode electrode). Theprotective layer 60 and the counter electrode 24 (or the first portion24A of the counter electrode 24) can have different standard reductionpotentials.

In the present disclosure, the potential difference (E_(cell) ⁰) betweenthe protective layer 60 and the counter electrode 24 is greater than 0.According to the Gibbs Free Energy relationship, i.e., ΔG⁰=−nFE_(cell)⁰, where ΔG⁰ is the change in the free energy, n is the mole number ofelectrons, and F is the charge per mole. When the Gibbs free energyΔG⁰<0, the reaction is a spontaneous reaction. From the above, it can beseen that when two oxidizable/reduceable substances with a potentialdifference (E_(cell) ⁰) are in the same reaction tank, the one withhigher standard reduction potential will tend to undergo reductionreaction and the other one will tend to undergo oxidation reaction. Forexample, when the standard reduction potential of the anode is smallerthan that of the cathode, the anode will spontaneously transferelectrons to the cathode, and the cathode will remain in the reducedstate because it continues to receive electrons, thus avoiding theinfluence of environmental oxidants (e.g., oxygen, water vapor, etc.).

The protective layer 60 and the counter electrode 24 are in the samereaction tank. In some embodiments, the protective layer 60 and thecounter electrode 24 are in contact with the air at the same time;however, the present disclosure is not limited thereto. The protectivelayer 60 is electrically connected to the counter electrode 24 and henceit is also considered to be in the same reaction tank. Since there isthe potential difference (E_(cell) ⁰) between the protective layer 60and the counter electrode 24, and the potential difference (E_(cell) ⁰)is greater than 0, an electron flow in a specific direction is generatedspontaneously, thereby allowing the subject to be protected (the counterelectrode 24 or the first portion 24A of the counter electrode 24) to bekept in the original oxidated/reduced status. In this way, one canprotect the biochemical test chip 300 to delay the unexpected spoilagedue to the reaction between the biochemical test chip 300 andenvironment.

In some embodiments, the area of the protective layer 60 is greater thanthe area of the counter electrode 24 (or the first portion 24A of thecounter electrode 24). In some embodiments, the area of the protectivelayer 60 substantially equals to the area of the counter electrode 24(or the first portion 24A of the counter electrode 24). The area andthickness of the protective layer 60 and the counter electrode 24 (orthe first portion 24A of the counter electrode 24) can be adjusteddepending on the system requirements. Depending on the materials of theprotective layer 60 and the counter electrode 24 (or the first portion24A of the counter electrode 24), the protective layer 60 and thecounter electrode 24 (or the first portion 24A of the counter electrode24) can, respectively, be the anode and the cathode, and the protectivelayer 60 and the counter electrode 24 (or the first portion 24A of thecounter electrode 24) can also be the cathode and the anode,respectively.

For simplification, as an example, the material of the first portion 24Aof the counter electrode 24 is silver (Ag); however, the presentdisclosure is not limited thereto. In the present embodiment, the firstportion 24A of the counter electrode 24 includes silver. However,silver, when exposed to the air, tends to react with oxygen and watervapor and becomes silver oxide, wherein the oxidation equation can beexpressed as 4Ag+O₂→2Ag₂O, wherein the standard reduction potential ofsilver/silver oxide is 1.17 V. When silver is oxidized into silver oxideafter being exposed to the air, it will result in the toxification onthe surface of the first portion 24A of the counter electrode 24. Inthis case, the capability of the first portion 24A to receive or releaseadditional electrons will be decreased, and therefore, the bottleneckeffect between the working electrode 22 and the counter electrode 24cannot be improved effectively.

As shown in FIG. 5 , the biochemical test chip 300 is disposed with theprotective layer 60, and the protective layer 60 is electricallyconnected to the counter electrode 24. In some embodiments, theprotective layer 60 is configured to protect the first portion 24A ofthe counter electrode 24. In some embodiments, the protective layer 60can include stannous oxide (SnO). Stannous oxide, when being exposed tothe air, tends to undergo oxidation reaction with water vapor, whereinthe reaction equation can be expressed as SnO+H₂O→SnO₂+2H⁺+2e⁻. Thestandard reduction potential of silver oxide/silver is 1.17 V, whereasthe standard reduction potential of stannous oxide/tin oxide −0.09 V.Hence, in the present embodiment, the first portion 24A of the counterelectrode 24 is the cathode, and the protective layer 60 is the anode.When the biochemical test chip 300 is exposed to environment with watervapor, the potential difference (E_(cell) ⁰) is 1.08 V. Because thepotential difference (E_(cell) ⁰) between the two is greater than 0, thechange in the free energy is smaller than 0, and hence, the followingreaction will vary out spontaneously: Ag₂O+SnO→2Ag+SnO₂. In this case,the half-reaction taking place on the first portion 24A of the counterelectrode 24 is Ag₂O+2H⁺+2e⁻ →2Ag+H₂O.

Therefore, silver oxide in the first portion 24A of the counterelectrode 24 is reduced into silver as a result of the oxidationreaction of the stannous oxide in the protective layer 60. When theprotective layer 60 undergoes oxidation reaction, the first portion 24Aof the counter electrode 24 undergoes the reduction reaction, therebyslowing the oxidation reaction caused by oxygen and water vapor in theair. Further, through the oxidation reaction between the water vapor inthe air and stannous oxide, the first portion 24A of the counterelectrode 24 is further protected, so as to keep the first portion 24Aof the counter electrode 24 stable. Therefore, by disposing theprotective layer 60 in the biochemical test chip 300, one can preventthe first portion 24A of the counter electrode 24 from being spoiledbefore performing the specimen measurement. The compositions andmaterials of the first portion 24A of the counter electrode 24 and theprotective layer 60 are not limited to those described above. In someembodiments, the compositions and materials of the first portion 24A ofthe counter electrode 24 and the protective layer 60 are chosen so thatthe potential difference (E_(cell) ⁰) between the two is greater than 0.

The present embodiment provides a biochemical test chip 300 with aprotective layer 60, wherein the protective layer 60 can maintain thestability of the active material in the counter electrode 24, so as toprotect the biochemical test chip 300 and slow the unexpected spoilageof the biochemical test chip 300 as a result of the reaction withenvironment before the biochemical test chip 300 is used in specimenmeasurement, thereby maintain or protecting the capability of thecounter electrode 24 of the biochemical test chip 300 in receiving orreleasing additional electrons.

FIG. 6 is a partial top view illustrating a biochemical test chipaccording to some embodiments of the present disclosure. As shown inFIG. 6 , the difference between the biochemical test chip 400 and thebiochemical test chip 100 is that the working electrode 22 and thecounter electrode 24 are compound electrode. The working electrode 22includes a first portion 22X and a second portion 22Y. The counterelectrode 24 includes a first portion 24X and a second portion 24Y. Insome embodiments, the working electrode 22 and the counter electrode 24are both made of an inactive material and an active material. Forexample, in the present embodiment, the first portion 22X of the workingelectrode 22 and the first portion 24X of the counter electrode 24include an inactive material, whereas the second portion 22Y of theworking electrode 22 and the second portion 24Y of the counter electrode24 include an active material.

In some embodiments, the inactive material can include carbon, and theactive material can include silver. The working electrode 22 and thecounter electrode 24 are composed of an inactive material and an activematerial to increase the overall electrical conductivity andconductivity. The first portion 22X of the working electrode 22completely overlaps with the second portion 22Y of the working electrode22, whereas the first portion 24X of the counter electrode 24 does notoverlap entirely with the second portion 24Y of the counter electrode24. In the present embodiment, the first portion 24X of the counterelectrode 24 at least partially exposes the second portion 24Y of thecounter electrode 24.

In some embodiments, the opening 32 at least partially exposes thesecond portion 24Y of the counter electrode 24, whereas the opening 32does not expose the second portion 22Y of the working electrode 22.Since the second portion 24Y of the counter electrode 24 includes anactive material, it has the capability to receive or release additionalelectrons. The second portion 24Y of the counter electrode 24 canreceive or release additional electrons from the biochemical test chip400 during the measurement reaction by being exposed through the opening32. In this way, the counter electrode 24's capability in maintainingthe electrical neutrality can be improved.

In some embodiments, the working electrode 22 and the counter electrode24 of the present embodiment can be formed by first disposing an activematerial on the insulating substrate 10 and then covering an inactivematerial on a predetermined position. The techniques for forming theworking electrode 22 and the counter electrode 24 can include screenprinting, imprinting, thermal transfer printing, spin coating, ink-jetprinting, laser ablation, deposition, electrodeposition, etc.; however,the present disclosure is not limited thereto.

FIG. 7 is a schematic exploded view illustrating a biochemical test chipaccording to some embodiments of the present disclosure. As shown inFIG. 7 , the difference between the biochemical test chip 500 and thebiochemical test chip 200 is that the counter electrode 24 is in afork-shape. The counter electrode 24 includes a first portion 24A and asecond portion 24B. In some embodiments, the first portion 24A and thesecond portion 24B are respectively made of an active material and aninactive material. The opening 32 at least partially exposes the firstportion 24A and the second portion 24B. In the opening 32, the counterelectrode 24 comprises both the active material (the first portion 24A)and the inactive material (the second portion 24B). In the presentembodiment, the second portion 24B of the counter electrode 24 isresponsible for the conventional task between the counter electrode 24and the matching working electrode 22, whereas the first portion 24A ofthe counter electrode 24 utilize its self-oxidation or self-reduction tocompensate for the insufficiency in the second portion 24B. In detail,the first portion 24A of the counter electrode 24 can receive or releaseadditional electrons from the biochemical test chip 500 duringmeasurement reaction.

FIG. 8 is a partial top view illustrating a biochemical test chipaccording to some embodiments of the present disclosure. As shown inFIG. 8 , the difference between the biochemical test chip 600 and thebiochemical test chip 200 is that the biochemical test chip 600 isdisposed with a first counter electrode 24 and a second counterelectrode 26, wherein the first counter electrode 24 and the secondcounter electrode 26 are separated from each other. The first counterelectrode 24 and the second counter electrode 26 respectively include afirst portion 24A, 26A and a second portion 24B, 26B. The first portion24A of the first counter electrode 24 and the first portion 26A of thesecond counter electrode 26 include an active material, whereas thesecond portion 24B of the first counter electrode 24 and the secondportion 26B of the second counter electrode 26 include an inactivematerial. The opening 32 at least partially exposes the first portion24A of the first counter electrode 24 and the first portion 26A of thesecond counter electrode 26.

In some embodiments, the material of the first portion 24A of the firstcounter electrode 24 can undergo self-oxidation reaction, and thematerial of the first portion 26A of the second counter electrode 26 canundergo self-reduction reaction. In some embodiments, the standardreduction potential of the first counter electrode 24 is greater thanthe standard reduction potential of the second counter electrode 26. Insome embodiments, the standard reduction potential of the first portion24A of the first counter electrode 24 is greater than the standardreduction potential of the first portion 26A of the second counterelectrode 26.

In some embodiments, the area of the first portion 24A of the firstcounter electrode 24 and the area of the first portion 26A of the secondcounter electrode 26 are is smaller than or equal to the area of theworking electrode 22. In some embodiments, the sum of the areas of thefirst portion 24A of the first counter electrode 24 and the firstportion 26A of the second counter electrode 26 that are exposed in theopening 32 is smaller than or equal to the area of the working electrode22 that is exposed in the opening 32.

The first portion 24A of the first counter electrode 24 and the firstportion 26A of the second counter electrode 26 are configured to receiveor release additional electrons from the biochemical test chip 600during measurement reaction by being exposed in the opening 32. Byswitching the electrical properties of the first counter electrode 24and the second counter electrode 26, no bottleneck effect occurs ineither the oxidation or reduction reactions of the working electrodes22. In this way, the biochemical chip 600 can measure the concentrationof substances under different reactions.

FIGS. 9A and 9B are charts respectively showing the signals detected ona counter electrode according to the present embodiment and a counterelectrode of a comparative example under different concentrations,wherein FIG. 9A shows the blood specimen signal of 43% hematocrit 200mg/dL blood glucose, and FIG. 9B shows the blood specimen signal of 43%hematocrit 600 mg/dL blood glucose. In detail, curve A of FIG. 9A andFIG. 9B shows the blood sample signal of a 4.8 mm²-working electrodewith a 0.8 mm²-counter electrode of the disclosed embodiment. Curve B isthe blood sample signal of the 4.8 mm²-working electrode with the 2.4mm²-counter electrode of the comparative example. The working electrodeis a carbon electrode. The 0.8 mm²-counter electrode of the embodimentsof the present disclosure is a silver oxide electrode; however, thepresent disclosure is not limited thereto. The 2.4 mm²-counter electrodeof the comparative example is a commercially available 2.4 mm²-carbonelectrode. Curve A and B show the comparison of the signals using theoxidation concentration measurement method under the same environmentalconditions.

As shown in FIG. 9A, since the general counter electrode (inactivematerial) is sufficient to support the amount of charge transfer for thereduction reaction on the working electrode in a low concentrationenvironment, the signal and performance of the counter electrode of thepresent embodiment and the counter electrode of the comparative exampleare almost the same, except for the difference in impedance obtainedbetween Curve A and Curve B.

As shown in FIG. 9B, in a high concentration environment, a bottleneckeffect occurs because the general counter electrode (inactive material)cannot match the charge transfer amount of the working electrode at highconcentration when the reduction reaction occurs. On the other hand,since the counter electrode of the present embodiment is capable ofself-oxidation and self-reduction, it can receive or release additionalelectrons, so the counter electrode of the present embodiment canmeasure higher signals using the same area or smaller area compared to aconventional electrode.

FIG. 10 is a chart showing the signals detected on a counter electrodeaccording to the present embodiment and a counter electrode of acomparative example having different electrode areas. FIG. 10 shows acomparison of the signals of the reduction concentration measurementmethod for the high concentration test using the counter electrode ofthe present embodiment and the counter electrode of the comparativeexample for a 600 mg/dL blood glucose plasma sample. The workingelectrode is a carbon electrode with an electrode area of 4.8 mm², andthe counter electrode of the present embodiment is a silver electrodewith an electrode area of 0.8 mm² (curve A), 1 mm² (curve B), and 1.2mm² (curve C). The counter electrodes of the comparative example arecarbon electrodes with electrode areas of 1.2 mm² (curve D), 1.8 mm²(curve E), and 2.4 mm² (curve F), respectively.

As shown in FIG. 10 , the intensity of the response current of thecomparative example increases as the area of the counter electrodeincreases, which means that the area of the counter electrode of thecomparative example is still insufficient to support the electron flowthrough the working electrode and thus creates a bottleneck effect.However, in the present embodiment, the current density of counterelectrode area does not increase as the area of the counter electrodeincreases, but remains a stable value. Therefore, the counter electrodeof this embodiment can be used with an electrode area smaller than thatof the counter electrode of the comparative example, and such electrodearea is sufficient to support the electron flow through the workingelectrode, so that there is no bottleneck effect.

It should be noted that there is a significant signal difference betweenthe counter electrode of the present embodiment and the counterelectrode of the comparative example. Under the high concentrationsituation, the working electrode has a large amount of trivalent ironions (Fe³⁺) for its electro-reduction. In contrast, the counterelectrode of the comparative example does not oxidize the same amount ofconductive dielectric at the same time, so the signal obtained is lessthan that of the counter electrode of the present example. The counterelectrode of the present embodiment is self-oxidizable and can releaseelectrons by oxidizing itself. In addition, the counter electrode of thepresent embodiment can also oxidize bivalent iron ions (Fe²⁺). In thisway, it is possible to provide a flow of electrons sufficient to matchthe flow of electrons on the working electrode, so that there is nobottleneck effect.

FIG. 11A and FIG. 11B are charts respectively showing the signalsdetected on an anode counter electrode and a cathode counter electrodewith different materials. The working electrode is a carbon electrode,the counter electrodes are made of three different materials, includingsilver (Curve A), silver oxide (AgO) (Curve B) and carbon (Curve C),respectively, and the same chemical condition is used to measure thecurrent density of (A/m²) of the biochemical test chip.

As shown in FIG. 11A, the counter electrode in FIG. 11A is the anode.Carbon cannot undergo self-oxidization or self-reduction, so the levelof the signal is related to the reactive layer and the area. Silver canundergo self-oxidization, and when the system environment satisfies theequation of E_(s) ⁰<E_(m) ⁰−E_(v), silver is self-oxidized, and hence,its current density is greater than that of the carbon. Silver oxidecannot undergo self-oxidization; hence, when the counter electrode isthe anode, even when the system environment satisfies the equation ofE_(s) ⁰<E_(m) ⁰−E_(v), it is not possible to oxidize silver oxide toprovide electrons, and hence, its current density (A/m²) is similar tothat of the carbon electrode.

It should be noted that the active material of the present disclosurerefers to the material that undergoes a secondary reaction when thecounter electrode is an anode and the system satisfies the equation ofE_(s) ⁰<E_(m) ⁰−E_(v); however, the present disclosure does not limit tothe embodiments where the secondary reaction can increase the currentdensity. In some embodiments, the current density of the counterelectrode is greater than twice of the current density of the workingelectrode. Moreover, the present disclosure does not limit the conditionof the counter electrode when in use; for example, the counter electrodemay comprise the active material as manufactured or may possess thefunctionality of the active material through the measuring apparatus. Insome embodiments, the counter electrode can be silver oxide, and afterthe specimen is loaded into the reaction zone, an appropriate thepotential is applied to reduce the silver oxide into silver.

As shown in FIG. 11B, the counter electrode in FIG. 11B is a cathode. Inthe present embodiment, silver without reduction capability cannotreceive additional electrons, and hence it cannot undergo the secondaryreaction when the system environment satisfies the condition of E_(s)⁰>E_(m) ⁰−E_(v), so the current density (A/m2) of silver is similar tothat of carbon. In contrast, silver oxide has reduction capability andcan undergo the secondary reaction when the system environment satisfiesthe condition of E_(s) ⁰>E_(m) ⁰−E_(v), and hence, the current density(A/m2) of silver oxide is higher than that of silver.

As discussed above, the active material of the present disclosure refersto materials capable of undergoing secondary reaction in a conditionmatching the polarity of the counter electrode. In other words, eventhough the active material can be self-oxidized or self-reduced underspecific environment, when the primary reaction is taking place, if theenvironment of the primary reaction cannot oxidize or reduce the activematerial, then such active material is not suitable to be used as thecounter electrode of the present disclosure.

The above description of the present disclosure provides a variety ofbiochemical test chips with a counter electrode including an activematerial, including a counter electrode with an active material that canprovide an amount of electrons equivalent to the amount of electronsgenerated by the conductive medium reaction on the working electrodewhen the electrode area is limited or the concentration of conductivemedium on the surface of the counter electrode in the reaction solutionis not high, and a suitable voltage is applied to the system, therebyenhancing the electroneutrality of the counter electrode and stabilizingthe electrochemical circuit without current bottleneck effects. In someembodiments, the biochemical test chip further includes a protectivelayer to aid in the stability of the active material on the electrode,thereby protecting the biochemical test chip and slowing or avoidingunintended spoilage of the biochemical test chip due to reaction withthe environment.

FIG. 12 is a schematic diagram illustrating an electrochemical system1000 according to some embodiments of the present disclosure. As shownin FIG. 12 , the electrochemical system 1000 includes an electrode unit1001 and a reactive unit 1002 electrically coupled to the electrode unit1001. In some embodiments, the electrode unit 1001 includes a workingelectrode and a counter electrode. Alternatively or additionally, theelectrode unit 1001 includes a spare electrode. As will be discussed ingreater detail below (i.e., in reference to FIGS. 13 to 15 ), in someembodiments, the reactive unit 1002 and a target analyte undergo aprimary reaction, and the counter electrode is configured to undergo asecondary reaction, wherein the secondary reaction does not interferewith the primary reaction, and the secondary reaction allows the counterelectrode to receive or release additional electrons.

As will be discussed in greater detail below (i.e., in reference toFIGS. 13 to 15 ), a current density of the counter electrode is greaterthan a current density of the working electrode. In some embodiments,the current density of the counter electrode is greater than or equal totwice the current density of the working electrode. In some embodiments,an area of the counter electrode is smaller than or equal to an area ofthe working electrode. In some embodiments, the counter electrode is acathode, and a standard reduction potential of an active material of thecounter electrode satisfies E_(s) ⁰>E_(m) ⁰−E⁰, where E_(s) ⁰ is astandard reduction potential of the active material, E_(m) ⁰ is astandard reduction potential for a concentration reaction on the workingelectrode, and E_(v) is a potential applied by a measuring apparatuswhile providing a measuring reaction. In some alternative embodiments,the counter electrode is an anode, and a standard reduction potential ofan active material of the counter electrode satisfies E_(s) ⁰<E_(m)⁰−E_(v), where E_(s) ⁰ is a standard reduction potential of the activematerial, E_(m) ⁰ is a standard reduction potential for a concentrationreaction on the working electrode, and E_(v) is a potential applied by ameasuring apparatus while providing a measuring reaction.

Alternatively or additionally, the electrochemical system 1000 includesa protective unit 1003. In some embodiments, the protective unit 1003 iselectrically coupled to the electrode unit 1001. As will be discussed ingreater detail below (i.e., in reference to FIGS. 22 to 24 ), theprotective unit 1003 is configured to oxidate the electrode unit 1001after the electrode unit 1001 receives an electron or to reduce theelectrode unit 1001 after the electrode unit 1001 loses an electron,wherein there is a potential difference (E_(cell) ⁰) between theprotective unit 1003 and the electrode unit 1001. In some embodiments,the potential difference (E_(cell) ⁰) is greater than 0.

FIG. 13 is a schematic view illustrating an implantable biochemical testchip 2000 according to some embodiments of the present disclosure. Theimplantable biochemical test chip 2000 may include one or more elementsof the electrochemical system 1000 as discussed above. As shown in FIG.13 , the implantable biochemical test chip 2000 includes a workingelectrode 2102, a counter electrode 2104, a reactive layer 2106, aninsulating layer 2108 and a biocompatible coating 2110. The workingelectrode 2102 and the counter electrode 2104 may together be referredto as the electrode unit 1001 of the electrochemical system 1000.Additionally, the reactive layer 2106 may be referred to as the reactiveunit 1002 of the electrochemical system 1000.

The implantable biochemical test chip 2000 may have a first end (ormeasuring end)2112 connected to a measuring apparatus and a second end(or implantable end) 2114 implanted into a specimen. It should be notedthat the implantable end 2114 is covered by the biocompatible coating2110. For clarity of discussion, the biocompatible coating 2110illustrated in FIG. 13 is shown in broken lines to show the componentscovered (i.e., the working electrode 2102, the counter electrode 2104,the reactive layer 2106 and the insulating layer 2108). In someembodiments, the implantable biochemical test chip 2000 is an elongatedstructure. Alternatively or additionally, the implantable biochemicaltest chip 2000 is a laminated structure.

FIG. 14A is a cross-sectional view of a structure 2102E including theworking electrode 2102 taken along line A-A′ of FIG. 13 according tosome embodiments of the present disclosure. As shown in FIG. 14A, thestructure 2102E includes the working electrode 2102, a substrate 2103and the reactive layer 2106.

FIG. 14B is a cross-sectional view of a structure 2104E including thecounter electrode 2104 taken along line B-B′ of FIG. 13 according tosome embodiments of the present disclosure. As shown in FIG. 14B, thestructure 2104E includes the working electrode 2102, the substrate 2103,the counter electrode 2104, the reactive layer 2106, the insulatinglayer 2108 and the biocompatible coating 2110.

Referring to FIGS. 13, 14A and 14B, in some embodiments, the substrate2103 is configured as a supporting base of the implantable biochemicaltest chip 2000. The substrate 2103 includes a material with certainmechanical strength and anti-buffering properties. In some embodiments,the substrate 2103 is electrically insulated. In some embodiments, amaterial of the substrate 2103 may include polyvinyl chloride (PVC),glass fiber (FR-4), polyester, polyethersulfone (PES), polyurethane(PU), polyether, polyamide (PA), polyimide (PI), bakelite, polyethyleneterephthalate (PET), polycarbonate (PC), polypropylene (PP),polyethylene (PE), polystyrene (PS), glass plate, ceramic, anycombination of the above-mentioned materials, or other suitablematerials; however, the present disclosure is not limited thereto. Thematerial of the substrate 2103 can be adjusted depending on the systemor actual needs.

In some embodiments, electrodes including the working electrode 2102 andthe counter electrode 2104 may be positioned at any specific locationsusing any suitable processes, such as chemical vapor deposition (CVD),physical vapor deposition (PVD), sputtering, reactive sputtering,printing, ablation (e.g., laser ablation), coating, dip coating,etching, the like, applied or otherwise treated. Each of the electrodes(including the working electrode 2102 and the counter electrode 2104)can include any conductive material. The conductive material mayinclude, but is not limited to, one or more of palladium (Pd), platinum(Pt), gold (Au), titanium (Ti), carbon (C), silver (Ag), copper (Cu),aluminum (Al), gallium (Ga), indium (In), iridium (Ir), iron (Fe), lead(Pb), magnesium (Mg), nickel (Ni), molybdenum (Mo), osmium (Os), rhodium(Rh), tin (Sn), zinc (Zn), silicon (Si), cobalt (Co), mercury (Hg),niobium (Nb), rhenium (Re), selenium (Se), tantalum (Ta), tungsten (W),uranium (U), vanadium (V), zirconium (Zr) or any combination of theabove conductive materials or their elemental derivatives. The presentdisclosure does not limit the relationship (e.g., spatial relationship)between the substrate 2103 and the working electrode 2102. In someembodiments, the implantable biochemical test chip 2000 uses aconductive material with a certain mechanical strength such that theworking electrode 2102 may replace the substrate 2103 and act as asubstrate of the implantable biochemical test chip 2000.

In some embodiments, the reactive layer 2106 may partially cover orentirely cover the working electrode 2102, wherein the reactive layer2106 is subjected to an electrochemical reaction at the implantable end2114. The reactive layer 2106 may include an enzyme and a conductivemedium. The enzyme may be highly specific to the specimen (or ananalysis target). The enzyme may be configured to undergo anelectrochemical reaction with the specimen (e.g., an analysis target,blood specimen or the like). The enzyme may include an immobilizedenzyme or an unimmobilized enzyme, such as redox enzymes, antigens,antibodies, microbial cells, animal or plant cells, or biologicallyidentifiable components of animal and plant tissues. The conductivemedium is configured to receive electrons generated after the reactionbetween the enzyme and the specimen and transmit the electrons to themeasuring apparatus via the working electrode 2102. In some embodiments,the conductive medium may be organic, organometallic, or inorganic,including but not limited to potassium hexacyanoferrate(III), potassiumhexacyanoferrate(II) trihydrate, ferric ferrocyanide, ruthenium complex,ferrocene, sodium dithionite, nicotinamide adenine dinucleotide (NAD⁺),nicotinamide adenine dinucleotide phosphate (NADP⁺), thiaminpyrophosphate (TPP), coenzyme A (HSCoA), flavin adenine dinucleotide(FAD) or a combination thereof, however, the present disclosure is notlimited thereto. In some embodiments, the reactive layer 2106 may befurther supplemented with a phosphate buffer and protectants, such asprotein, dextrin, glucan, amino acid, etc.; however, the presentdisclosure is not limited thereto. In some embodiments, the workingelectrode 2102 and part of the reactive layer 2106 (e.g., the conductivemedium) are components of a composite. For example, a conductive pasteis mixed with the conductive medium and then printed on the substrate2103 to form the working electrode 2102.

Referring to FIG. 14B, in some embodiments, the insulating layer 2108 isused to electrically insulate the working electrode 2102 from thecounter electrode 2104 before the implantable biochemical test chip 2000is brought into contact with the specimen (i.e., before implantation).The insulating layer 2108 may be any insulating material, for examplebut not limited to polyurethane, parylene, polyimide,polydimethylsiloxane (PDMS), liquid crystal polymer (LCP), PVCinsulating tape, PET insulating tape, thermal drying insulating paint,UV light-curing insulating paint, or photoresist. The counter electrode2104 is disposed over the insulating layer 2108.

In some embodiments of the present disclosure, the working electrode2102 and the counter electrode 2104 are formed by stacking in sequence.Both the counter electrode 2104 and the working electrode 2102 areexposed at the implantable end 2114 to form an electrical loop with thespecimen. For example, the specimen may include blood, tissue, fluid,and other biological samples; however, the present disclosure is notlimited thereto. In addition, both the counter electrode 2104 and theworking electrode 2102 are exposed at the measuring end 2112 and of acertain size to form an electrical loop with the measuring apparatus.

In some alternative embodiments, in order to improve thebiocompatibility of the implantable biochemical test chip 2000 with aliving body, the biocompatible coating 2110 is provided on theimplantable end 2114. The biocompatible coating 2110 is apolymer-blocking membrane configured to prevent or reduce penetration ofbiomolecules (such as white blood cells, proteins, fibroblasts, bloodclots, etc.) into an electrode surface (e.g., surfaces of the workingelectrode 2102 or the counter electrode 2104). The biocompatible coating2110 may be further configured to prevent or reduce the penetration byor adhesion of biomolecules to the biocompatible coating 2110 that couldchange electrical properties of the implantable biochemical test chip2000. In addition, the biocompatible coating 2110 may be configured toprovide small molecule analytes to penetrate through and reach a surfaceof an electrode (e.g., surfaces of the working electrode 2102 or thecounter electrode 2104) and react with the electrode to generate adetectable current. The polymer-blocking membrane may be hydrogel,chitin or its derivatives, hyaluronic acid or its derivatives,polyurethane, polyethylene-copolytetrafluoroethylene, polypropylene,polyvinylchloride, polyvinylidene fluoride, polybutylene terephthalate,polymethylmethacrylate, polyether ether ketone, cellulose ester,polysulfone, polyolefin, polyester, polycarbonate, silicone,polyethylene, polypropylene, nylon, polyacrylonitrile,polytetrafluoroethylene, expanded polytetrafluoroethylene or acombination thereof. In some embodiments, the biocompatible coating 2110may be formed by layering hydrophilic and hydrophobic materials.

FIG. 15 is a schematic diagram illustrating an electrochemical reactionof the electrochemical system 1000 or the implantable biochemical testchip 2000 according to some embodiments of the present disclosure. Forease of understanding, FIG. 15 illustrates only a portion of theelectrochemical system 1000 or the implantable biochemical test chip2000. In detail, FIG. 15 illustrates only portions of the workingelectrode 2102 and the counter electrode 2104 within a reaction zone(i.e., at the implantable end 2114). In addition, FIG. 15 furtherillustrates portions of an enzyme E and a conductive medium C within thereaction zone, wherein the enzyme E and the conductive medium C form aportion of the reactive layer 2106.

The working electrode 2102 allows a target analyte A to undergo theelectro-oxidation reaction or electro-reduction reaction on the surfaceof the working electrode 2102. The working electrode 2102 can be used bythe measuring apparatus M to determine a concentration of the targetanalyte A. In detail, the electro-oxidation reaction or theelectro-reduction reaction is an electrochemical reaction in which thetarget analyte undergoes an exchange between electrical and chemicalenergy on the surface of the working electrode 2102. The workingelectrode 2102 can be an anode or a cathode, depending on a requirementof the measurement reaction. For example, if the target analyte A isoxidized on the working electrode 2102, the working electrode 2102 is ananode; if the target analyte A is reduced on the working electrode 2102,the working electrode 2102 is a cathode. The counter electrode 2104 isan electrode that undergoes the electro-reduction reaction or theelectro-oxidation reaction corresponding to the working electrode 2102so that the overall electrochemical system satisfies principles ofcharge balance. A potential and a polarity of the counter electrode 2104are opposite to those of the working electrode 2102. Before coming intocontact with the specimen S, the working electrode 2102 and the counterelectrode 2104 are insulated from each other. After the workingelectrode 2102 and the counter electrode 2104 are in contact with thespecimen S, they form the electrical loop with the measuring apparatusM.

In some embodiments, the specimen S includes the target analyte A,wherein the target analyte A includes an electrochemically activesubstance or an electrochemically reactive substance. After the specimenS is loaded into the reaction zone, the target analyte A in the specimenS is reduced by the enzyme E in the reactive layer 2106, thereby forminga reduced target analyte A′; however, the present disclosure is notlimited thereto. In other embodiments, the target analyte A in thespecimen S can be oxidized by the enzyme E in the reactive layer 2106,thereby forming an oxidized target analyte. The conductive medium C ofthe reactive layer 2106 is configured to receive or provide theelectrons generated or lost due to the reaction between the enzyme E andthe target analyte A in the specimen S and to transmit the electrons tothe measuring apparatus M via the working electrode 2102.

For ease of understanding, the conductive medium C in this embodimentincludes, for example, Fe²⁺; however, the present disclosure is notlimited thereto. When the target analyte A in the specimen S is reduced,the conductive medium C of the reactive layer 2106 is oxidized. In thepresent embodiment, Fe²⁺ is oxidized into Fe³⁺. Moreover, the conductivemedium C of the reactive layer 2106 is oxidized to an extentcorresponding to an extent to which the target analyte A is reduced.When the conductive medium C (e.g., Fe²⁺) releases electrons, theworking electrode 2102 also undergoes a reaction to reduce an oxidizedconductive medium C′ (e.g., Fe³⁺) into a reduced conductive medium C(e.g., Fe²⁺). The measuring apparatus M detects changes in a number ofelectrons (e⁻) generated by the reaction on the working electrode 2102and carries out a concentration analysis. When the working electrode2102 undergoes the reduction reaction, the counter electrode 2104 mustoxidize a corresponding amount of the reduced conductive medium C (e.g.,Fe²⁺), so that the overall reaction achieves charge balance.

Generally, when an oxidizability of the counter electrode 2104 isinsufficient to match that of the working electrode 2102; e.g., when theamount of the conductive medium C oxidized by the counter electrode 2104is smaller than an amount of the conductive medium C reduced by theworking electrode 2102, a level of a reducing current generated by theworking electrode 2102 is limited due to a principle of electricalneutrality, thereby incurring a bottleneck effect. The bottleneckeffect, caused by the working electrode 2102 reacting with greaterelectron flow than the counter electrode 2104 is able to react with,limits a range of a measurable concentration of the electrochemicalsystem 1000 or the implantable biochemical test chip 2000.

In some embodiments, a material of the counter electrode 2104 in thereaction zone is selected to have a better electrochemical reactivitywith environmental substances (i.e., substances that are not the targetanalyte A of the specimen S). In the present embodiment, the counterelectrode 2104 can include an active material (which is alsoconductive). A purpose of using the active material is that when theelectrochemical system 1000 or the implantable biochemical test chip2000 undergoes an electrochemical reaction, an active material of thecounter electrode 2104 can carry out its self-secondary redox reactionwithout interfering with a primary reaction. Such primary reactionrefers to the oxidation or the reduction reaction caused by the targetanalyte A and the reactive layer 2106, and the secondary redox reactionrefers to the oxidation or the reduction reaction that is not caused bythe target analyte A and the reactive layer 2106. In detail, as long asthe secondary redox reaction occurring on the counter electrode 2104does not affect the primary reaction of the working electrode 2102 orthe counter electrode 2104, the active material of the counter electrode2104 is not limited. In addition, materials required for the secondaryredox reaction can come from either the specimen S or the environment(e.g., human body tissue).

By having the active material in the counter electrode 2104, the counterelectrode 2104 is capable of providing, by its own secondary redoxreaction, an amount of electrons equivalent to that generated by thereaction of the conductive medium C on the working electrode 2102. Suchsecondary redox reaction allows the counter electrode 2104 to receive orrelease additional electrons. The additional electrons refer toelectrons not generated by the enzyme E or the conductive medium C ofthe reactive layer 2106. In other words, in addition to undergoing theelectrochemical reaction with the target analyte A in the specimen S,the counter electrode 2104 can obtain electrons by undergoing thesecondary redox reaction with the active material without disrupting theelectrical neutrality of the measuring apparatus M.

For example, after the specimen S is provided or deposited in thereaction zone and the measuring apparatus M supplies a working voltage,the target analyte A in the specimen S and the enzyme E of the reactivelayer 2106 undergo the reaction, and a corresponding amount of theoxidized conductive medium C′ on the working electrode 2102 is reducedto the reduced conductive medium C. On the other hand, the counterelectrode 2104 needs to oxidize the reduced conductive medium C to theoxidized conductive medium C′ to an extent that corresponds to a numberof reduced electrons of the working electrode 2102, in order to maintainthe electrical neutrality of the overall system. Since the counterelectrode 2104 of the present disclosure has an active material that canundergo the secondary redox reaction, when the counter electrode 2104oxidizes the reduced conductive medium C and receives electrons, it alsoperforms an oxidation reaction at the same time to generate additionalelectrons. The active material of the counter electrode 2104 can performits own oxidation or reduction reaction without interfering with theprimary reaction of the counter electrode 2104. By having the activematerial in the counter electrode 2104, the counter electrode 2104 iscapable of providing, by its own secondary redox reaction, an amount ofelectrons equivalent to that generated by the reaction of the conductivemedium C on the working electrode 2102 when the electrode area islimited or when the concentration of the conductive medium C on thesurface of the counter electrode 2104 in the reaction solution isinsufficient for electron transfer. As a result, an ability of thecounter electrode 2104 to achieve electrical neutrality can be improved,and the electrochemical circuit can be stabilized to avoid a currentbottleneck effect on the counter electrode 2104. In some embodiments,the secondary redox reaction of the active material and the primaryreaction of the counter electrode 2104 are both oxidation reactions.Alternatively, the secondary redox reaction of the active material andthe primary reaction of the counter electrode 104 are both reductionreactions.

For simplification, silver (Ag) is used as an example of the activematerial of the counter electrode 2104; however, the present disclosureis not limited thereto. In detail, silver (Ag) in the counter electrode2104 is in direct contact with the specimen S. In addition to theworking voltage supplied by the measuring apparatus M, the reaction zonefurther comprises water (H₂O) or hydroxide ions (OH⁻) that allow silver(Ag) to be oxidized. Therefore, under appropriate conditions, silver inthe counter electrode 2104 can undergo oxidation reaction with thehydroxide ions (OH⁻), water (H₂O) or water vapor from the specimen S orthe environment, and the reaction can be expressed by a formula:

2Ag+2OH⁻→Ag₂O+H₂O+2e⁻

or

2Ag+H₂O→Ag₂O+2H⁺+2e⁻.

Silver oxide (Ag₂O) and water (H₂O) are generated and electrons arereleased after the silver and hydroxide ions (OH⁻) or water from thespecimen S or the environment undergo the redox reaction. Therefore,with respect to the overall reaction, the counter electrode 2104 is alsoself-oxidized to form the oxidized electrode (silver oxide) and releaseelectrons (e⁻), in addition to oxidizing the reduced conductive medium C(e.g., Fe²⁺). Hence, electrons generated by the active material of thecounter electrode 2104 help to improve an overall capability (e.g.,electrical neutrality) of the counter electrode 2104 under a principleof conservation of charge.

Thus, when the working electrode 2102 undergoes electro-reduction, thecounter electrode 2104 can undergo electro-oxidation correspondingly tosatisfy the overall system's electrical neutrality. It should be notedthat although the active material (e.g., silver) of the counterelectrode 2104 consumes water (H₂O) or hydroxide ions (OH⁻) and produceshydrogen ions (H⁺) or water (H₂O) during the oxidation process, andthereby partially changes a pH of the reaction zone, the resultingchange in pH is negligible for the overall system. Hence, the pH changedoes not affect the primary reaction and the test results, and cantherefore be ignored.

To ensure that the active material of the counter electrode 2104 has theabove-mentioned capabilities, the active material and the counterelectrode 2104 must have a same reaction polarity. In other words,although a change in oxidation numbers of the active material and thecounter electrode 2104 may not be equal, the active material must haveoxidizing ability if the counter electrode 2104 is an anode, and theactive material must have reducing ability if the counter electrode 2104is a cathode. Therefore, the active material needs to match the reactionpolarity of the counter electrode 2104, and must be selected so as toimprove the overall capability (e.g., electrical neutrality) of thecounter electrode 2104 under the principle of conservation of charge.Therefore, when the counter electrode 2104 is an anode, a standardreduction potential of the active material of the counter electrode 2104must satisfy E_(s) ⁰<E_(m) ⁰−E_(v). On the other hand, when the counterelectrode 2104 is a cathode, the standard reduction potential of theactive material of the counter electrode 2104 must satisfy E_(s) ⁰>E_(m)⁰−E_(v). Here, E_(s) ⁰ is the standard reduction potential of the activematerial, E_(m) ⁰ is the standard reduction potential of theconcentration reaction on the working electrode 2102, and E_(v) is apotential supplied by the measuring apparatus M when providing ameasuring reaction.

In some embodiments, the conductive medium C is Fe^(III)(CN)₆ ⁴⁻, theworking voltage (E_(v)) supplied by the measuring apparatus M is −0.4V,the working electrode 2102 undergoes reduction reaction, and hence thereaction of the conductive medium C on the surface of the workingelectrode 2102 is Fe^(III)(CN)₆ ⁴⁻+e⁻ →Fe^(II)(CN)₆ ⁴⁻, wherein E_(m)⁰=0.36 V. Therefore, in the present embodiment, the counter electrode2104 is an anode. The standard reduction potential (E_(s) ⁰) of theactive material of the counter electrode 2104 undergoing the reductionreaction must be less than 0.76V; thus, a material with a standardreduction potential (E_(s) ⁰) less than 0.76V can be chosen as theactive material for the counter electrode 2104; such material mayinclude ferrocene (E⁰=+0.4V), Cu (E⁰=+0.34V), Fe (E⁰=+0.085V), Sn(E⁰=−0.1V), or the like; however, the present disclosure is not limitedthereto.

In some embodiments, the conductive medium C is Fe^(II)(CN)₆ ⁴⁻, theworking voltage (E_(v)) supplied by the measuring apparatus M is +0.4V,the working electrode 2102 undergoes oxidation reaction, and hence thereaction of the conductive medium C on the surface of the workingelectrode 2102 is Fe^(II)(CN)₆ ⁴⁻→Fe^(III)(CN)₆ ³⁻+e⁻, wherein E_(m)⁰=0.36V. Therefore, in the present embodiment, the counter electrode2104 is a cathode. The standard reduction potential (E_(s) ⁰) of theactive material of the counter electrode 2104 undergoing the reductionreaction must be greater than −0.04V; thus, a material with a standardreduction potential (E_(s) ⁰) greater than −0.04V can be chosen as thematerial for the counter electrode 2104; such material may include Fe₃O₄(E⁰=+0.085V), AgCl (E⁰=+0.2223V), ferrocenium (E⁰=+0.4V), benzoquinone(E⁰=+0.6992V) or the like; however, the present disclosure is notlimited thereto.

The foregoing is only an example of the active material of the counterelectrode 2104, and the present disclosure is not limited thereto.Moreover, the standard reduction potential of the active material of thecounter electrode 2104 is not limited to the foregoing examples. Asdiscussed above, one should consider the polarity of the active materialof the counter electrode 2104, wherein the polarity of the activematerial should be same as a polarity of the measuring reaction on thecounter electrode 2104. Further, the standard reduction potential of theactive material of the counter electrode 2104 should satisfy thecondition of E_(s) ⁰<E_(m) ⁰−E_(v) or E_(s) ⁰>E_(m) ⁰−E_(v). In someembodiments, E_(v) can be between ±5 V and ±2 mV. In some embodiments,E_(v) can be between ±2 V and ±80 mV. In some embodiments, E_(v) can bebetween ±0.8 V and ±0.1 V.

In the present disclosure, the material of the working electrode 2102and the material of the counter electrode 2104 are particularly chosenso that the current density of the counter electrode 2104 is greaterthan the current density of the working electrode 2102. In someembodiments, the material of the counter electrode 2104 has betterelectrochemical reactivity compared with that of the working electrode2102. In detail, the material of the counter electrode 2104 is selectedto have a better electrochemical reactivity with environmentalsubstances (i.e., substances that are not the target analyte A of thespecimen S) compared to that of the working electrode 2102. In someembodiments, the area of the counter electrode 2104 may be smaller thanor equal to the area of the working electrode 2102. In some embodiments,the current density of the counter electrode 2104 is greater than orequal to two times the current density of the working electrode 2102.

In the present disclosure, the counter electrode 2104 can include anactive material which is also conductive. In some embodiments, theactive material may be doped in the counter electrode 2104. In someembodiments, the active material may be formed on the surface of thecounter electrode 2104. In some embodiments, the counter electrode 2104is composed of the active material. The active material of the counterelectrode 2104 refers to a substance that can undergo redox reactionwithin the working voltage range. The working voltage refers to thevoltage provided by the measuring apparatus M to cause the workingelectrode 2102 and the counter electrode 2104 to perform theelectrochemical reaction. The active material also refers to a materialthat is able to promote its own oxidation or reduction reaction under anelectrochemical testing environment. Material properties of the counterelectrode 2104 must be compatible with the working electrode 2102. Insome embodiments, the active material of the counter electrode 2104 caninclude, but is not limited to, silver (Ag), tin (Sn), iron (Fe), zinc(Zn), cobalt (Co), nickel (Ni), lead (Pb), copper (Cu), manganese (Mn),metal complexes thereof, or a combination thereof, however, the presentdisclosure is not limited thereto. In some alternative embodiments,depending on the system or actual needs, the material of the counterelectrode 2104 may be a mixture of active materials and theiroxidation/reduction states such as silver/silver chloride (Ag/AgCl).

The present disclosure is not limited to the foregoing embodiments, andcan comprise other different embodiments. For simplification purposesand to facilitate comparison between embodiments of the presentdisclosure, in the following embodiments, identical (or similar)elements are labeled with identical (or similar) reference numerals. Tofurther facilitate the comparison of the embodiments, only differencesamong embodiments are discussed, whereas identical (or similar) featuresare not discussed for the sake of brevity.

FIG. 16 is a schematic view illustrating an implantable biochemical testchip 3000 according to some embodiments of the present disclosure. Theimplantable biochemical test chip 3000 may include one or more elementsof the electrochemical system 1000 as discussed above. As shown in FIG.16 , the implantable biochemical test chip 3000 includes a workingelectrode 3102, a substrate 3103, a counter electrode 3104 and areactive layer 3106 (see FIG. 17 ). The implantable biochemical testchip 3000 may further include a spare electrode 3220. The workingelectrode 3102, the counter electrode 3104 and the spare electrode 3220may together be referred to as the electrode unit 1001 of theelectrochemical system 1000. Additionally, the reactive layer 3106 maybe referred to as the reactive unit 1002 of the electrochemical system1000.

The working electrode 3102, the counter electrode 3104 and the spareelectrode 3220 are arranged on one side (i.e., on a same side) of thesubstrate 3103. In some embodiments, the spare electrode 3220 may be areference electrode. Alternatively, the spare electrode 3220 may be asecond working electrode or a second counter electrode. In somealternative embodiments, the spare electrode 3220 may be a spare counterelectrode or a common counter electrode. In some embodiments, the spareelectrode 3220 may form another electrical loop with the workingelectrode 3102 or the counter electrode 3104 to perform a secondmeasurement, such as a measurement of a second concentration. The secondconcentration may be independent from a first concentration (e.g., aconcentration of the target analyte A). Alternatively, the secondconcentration may be used to correct or calibrate the firstconcentration. In some alternative embodiments, the second measurementmay be a non-concentration measurement (such as a detection forimplantation or a detection of withdrawal from implantation).

The implantable biochemical test chip 3000 may have a first end (ormeasuring end) 3112 connected to a measuring apparatus and a second end(or implantable end) 3114 implanted into a specimen. In someembodiments, a biocompatible coating 3110 (see FIG. 17 ) partiallycovers the implantable biochemical test chip 3000. For example, theimplantable end 3114 is covered by the biocompatible coating 3110.

FIG. 17 is a cross-sectional view illustrating the implantablebiochemical test chip 3000 taken along line C-C′ of FIG. 16 according tosome embodiments of the present disclosure. As shown in FIG. 17 , theimplantable biochemical test chip 3000 includes the working electrode3102, the substrate 3103, the counter electrode 3104, the reactive layer3106, the biocompatible coating 3110 and the spare electrode 3220. Thebiocompatible coating 3110 and the reactive layer 3106 coat the workingelectrode 3102, the spare electrode 3220, and the counter electrode 3104to provide an electrochemical environment required for measurement atthe implantable end 3114.

It should be noted that a spatial relationship between the workingelectrode 3102, the counter electrode 3104 and the spare electrode 3220can be adjusted depending on system requirements.

FIG. 18 is a schematic view illustrating an implantable biochemical testchip 4000 according to some embodiments of the present disclosure. Theimplantable biochemical test chip 4000 may include one or more elementsof the electrochemical system 1000 as discussed above. As shown in FIG.18 , the implantable biochemical test chip 4000 includes a workingelectrode 4102, a substrate 4103 and a counter electrode 4104.Additionally, the implantable biochemical test chip 4000 may furtherinclude a reactive layer (not shown). The working electrode 4102 and thecounter electrode 4104 may together be referred to as the electrode unit1001 of the electrochemical system 1000. Additionally, the reactivelayer may be referred to as the reactive unit 1002 of theelectrochemical system 1000. The implantable biochemical test chip 4000may have a first end (or measuring end) 4112 connected to a measuringapparatus and a second end (or implantable end) 4114 implanted into aspecimen.

FIG. 19 is a schematic exploded view illustrating an implantablebiochemical test chip 5000 according to some embodiments of the presentdisclosure. The implantable biochemical test chip 5000 may include oneor more elements of the electrochemical system 1000 as discussed above.As shown in FIG. 19 , the implantable biochemical test chip 5000includes a working electrode 5102, a substrate 5103, a counter electrode5104, a reactive layer 5106 (see FIG. 20 ) and a spare electrode 5220.The working electrode 5102, the counter electrode 5104 and the spareelectrode 5220 may together be referred to as the electrode unit 1001 ofthe electrochemical system 1000. Additionally, the reactive layer 5106may be referred to as the reactive unit 1002 of the electrochemicalsystem 1000.

The implantable biochemical test chip 5000 may have a first end (ormeasuring end) 5112 connected to a measuring apparatus and a second end(or implantable end) 5114 implanted into a specimen. As shown in FIG. 19, the counter electrode 5104 and the working electrode 5102 are arrangedat different sides of the substrate 5103. In some embodiments, the spareelectrode 5220 is arranged on a same side as the counter electrode 5104.Alternatively, the spare electrode 5220 is arranged on a same side asthe working electrode 5102. In some embodiments, a biocompatible coating5110 (see FIG. 20 ) partially covers the implantable biochemical testchip 5000. For example, the implantable end 5114 is covered by thebiocompatible coating 5110.

FIG. 20 is a cross-sectional view illustrating the implantablebiochemical test chip 5000 taken along line D-D′ of FIG. 19 according tosome embodiments of the present disclosure. As shown in FIG. 20 , theimplantable biochemical test chip 5000 includes the working electrode5102, the substrate 5103, the counter electrode 5104, the reactive layer5106, the biocompatible coating 5110 and the spare electrode 5220. Thereactive layer 5106 is disposed at least on one side of the workingelectrode 5102; however, the present disclosure is not limited thereto.A position of the reactive layer 5106 can be adjusted depending on asystem or actual needs.

FIG. 21 is a schematic exploded view illustrating an implantablebiochemical test chip 6000 according to some embodiments of the presentdisclosure. The implantable biochemical test chip 6000 may include oneor more elements of the electrochemical system 1000 as discussed above.As shown in FIG. 21 , the implantable biochemical test chip 6000includes a working electrode 6102, a substrate 6103 and a counterelectrode 6104. Additionally, the implantable biochemical test chip 6000may further include a reactive layer (not shown). The working electrode6102 and the counter electrode 6104 may together be referred to as theelectrode unit 1001 of the electrochemical system 1000. Additionally,the reactive layer may be referred to as the reactive unit 1002 of theelectrochemical system 1000. The implantable biochemical test chip 6000may have a first end (or measuring end) 6112 connected to a measuringapparatus and a second end (or implantable end) 6114 implanted into aspecimen. The counter electrode 6104 and the working electrode 6102 arearranged at different sides of the substrate 6103.

FIGS. 22 to 24 illustrate alternative embodiments of the presentapplication. In some embodiments, as shown in FIGS. 22 to 24 , a counterelectrode (e.g., 7104, 8104 or 9104) is provided with a protective layer(e.g., 7122, 8122 or 9122) at a measuring end (e.g., 7112, 8112 or9112). In some embodiments, the protective layer (e.g., 7122, 8122 or9122) is configured to further stabilize an active material on thecounter electrode (e.g., 7104, 8104 or 9104), thereby protecting animplantable biochemical test chip (e.g., 7000, 8000 or 9000) andmitigating or avoiding unexpected variations in the implantablebiochemical test chip (e.g., 7000, 8000 or 9000) and a surroundingenvironment. In some embodiments, the protective layer (e.g., 7122, 8122or 9122) is electrically connected to the counter electrode (e.g., 7104,8104 or 9104). In some embodiments, the protective layer (e.g., 7122,8122 or 9122) and the counter electrode (e.g., 7104, 8104 or 9104) arelocated at a same level. In some embodiments, the protective layer(e.g., 7122, 8122 or 9122) and the counter electrode (e.g., 7104, 8104or 9104) are located at different levels. For example, the protectivelayer (e.g., 7122, 8122 or 9122) can be disposed above or under thecounter electrode (e.g., 7104, 8104 or 9104). The protective layer(e.g., 7122, 8122 or 9122) can take forms of solid, liquid or gas. Forexample, solids can include pure metals, alloys, metal compounds(halides, oxidates, mixed-valence compounds, organometallic complexes),organic redox agents, or the like. Liquids can include aqueoussolutions, organic solutions, supercritical fluids, liquid elements(e.g., bromine, mercury), or the like. Gases can include gaseouselements (e.g., oxygen, ozone), gaseous compounds (e.g., ammonium,nitrogen monoxides), or the like.

FIG. 22 is a schematic view illustrating an implantable biochemical testchip 7000 according to some embodiments of the present disclosure. Theimplantable biochemical test chip 7000 may include one or more elementsof the electrochemical system 1000 as discussed above. As shown in FIG.22 , the implantable biochemical test chip 7000 includes a workingelectrode 7102, a counter electrode 7104, a reactive layer 7106, aninsulating layer 7108, a biocompatible coating 7110 and a protectivelayer 7122. The working electrode 7102 and the counter electrode 7104may together be referred to as the electrode unit 1001 of theelectrochemical system 1000. Additionally, the reactive layer 7106 maybe referred to as the reactive unit 1002 of the electrochemical system1000. Moreover, the protective layer 7122 may be referred to as theprotective unit 1003 of the electrochemical system 1000.

The implantable biochemical test chip 7000 may have a first end (ormeasuring end)7112 connected to a measuring apparatus and a second end(or implantable end) 7114 implanted into a specimen. The protectivelayer 7122 may be arranged over the counter electrode 7104 at themeasuring end 7112. Alternatively, the protective layer 7122 and thecounter electrode 7104 are located at a same level.

FIG. 23 is a schematic view illustrating an implantable biochemical testchip 8000 according to some embodiments of the present disclosure. Theimplantable biochemical test chip 8000 may include one or more elementsof the electrochemical system 1000 as discussed above. As shown in FIG.23 , the implantable biochemical test chip 8000 includes a workingelectrode 8102, a substrate 8103, a counter electrode 8104, a reactivelayer (not shown), a protective layer 8122 and a spare electrode 8220.The working electrode 8102, the counter electrode 8104 and the spareelectrode 8220 may together be referred to as the electrode unit 1001 ofthe electrochemical system 1000. Additionally, the reactive layer may bereferred to as the reactive unit 1002 of the electrochemical system1000. Moreover, the protective layer 8122 may be referred to as theprotective unit 1003 of the electrochemical system 1000. The implantablebiochemical test chip 8000 may have a first end (or measuring end) 8112connected to a measuring apparatus and a second end (or implantable end)8114 implanted into a specimen. The protective layer 8122 is arrangedadjacent to the counter electrode 8104 at the measuring end 8112.

FIG. 24 is a schematic exploded view illustrating an implantablebiochemical test chip 9000 according to some embodiments of the presentdisclosure. The implantable biochemical test chip 9000 may include oneor more elements of the electrochemical system 1000 as discussed above.As shown in FIG. 24 , the implantable biochemical test chip 9000includes a working electrode 9102, a substrate 9103, a counter electrode9104, a reactive layer (not shown), a protective layer 9122 and a spareelectrode 9220. The working electrode 9102, the counter electrode 9104and the spare electrode 9220 may together be referred to as theelectrode unit 1001 of the electrochemical system 1000. Additionally,the reactive layer may be referred to as the reactive unit 1002 of theelectrochemical system 1000. Moreover, the protective layer 9122 may bereferred to as the protective unit 1003 of the electrochemical system1000. The implantable biochemical test chip 9000 may have a first end(or measuring end) 9112 connected to a measuring apparatus and a secondend (or implantable end) 9114 implanted into a specimen. The protectivelayer 9122 is arranged adjacent to the counter electrode 9104 at themeasuring end 9112.

In some embodiments, there can be a potential difference (E_(cell) ⁰)between the protective unit 1003 and the electrode unit 1001. Forexample, there can be a potential difference (E_(cell) ⁰) between theprotective layer 7122, 8122 or 9122 and the counter electrode 7104, 8104or 9104. The potential difference (E_(cell) ⁰) can be expressed asE_(cell) ⁰=E_(cathode)−E_(anode), where E_(cathode) is a standardreduction potential of the cathode, and E_(anode) is a standardreduction potential of the anode. The protective layer 7122, 8122 or9122 and the counter electrode 7104, 8104 or 9104 can have differentstandard reduction potentials. In the present disclosure, the potentialdifference (E_(cell) ⁰) between the protective layer 7122, 8122 or 9122and the counter electrode 7104, 8104 or 9104 is greater than 0.According to a Gibbs Free Energy relationship, i.e., ΔG⁰=−nFE_(cell) ⁰,where ΔG⁰ is a change in free energy, n is a quantity of electrons inmoles, and F is a charge per mole. When ΔG⁰<0, a reaction is aspontaneous reaction. From the above, it can be seen that when twooxidizable/reduceable substances with a potential difference (E_(cell)⁰) are in a same reaction tank, a substance having a higher standardreduction potential will tend to undergo a reduction reaction andanother substance will tend to undergo an oxidation reaction. Forexample, when the standard reduction potential of the anode is smallerthan that of the cathode, the anode will spontaneously transferelectrons to the cathode, and the cathode will remain in a reduced statebecause it continues to receive electrons, thus avoiding an influence ofenvironmental oxidants (e.g., oxygen, water vapor, etc.).

The protective layer 7122, 8122 or 9122 and the counter electrode 7104,8104 or 9104 are in a same reaction tank. In some embodiments, theprotective layer 7122, 8122 or 9122 and the counter electrode 7104, 8104or 9104 are in contact with air at a same time; however, the presentdisclosure is not limited thereto. The protective layer 7122, 8122 or9122 is electrically connected to the counter electrode 7104, 8104 or9104 and hence the protective layer 7122, 8122 or 9122 is alsoconsidered to be in the same reaction tank. Since there is the potentialdifference (E_(cell) ⁰) between the protective layer 7122, 8122 or 9122and the counter electrode 7104, 8104 or 9104, and the potentialdifference (E_(cell) ⁰) is greater than 0, an electron flow in aspecific direction is generated spontaneously, thereby allowing anobject to be protected (i.e., the counter electrode 7104, 8104 or 9104)to be kept in an original oxidized/reduced status. In this way, theimplantable biochemical test chip 7000, 8000 or 9000 can be protected inorder to reduce unexpected spoilage due to reaction between theimplantable biochemical test chip 7000, 8000 or 9000 and theenvironment. An area and a thickness of the protective layer 7122, 8122or 9122 and the counter electrode 7104, 8104 or 9104 can be adjusteddepending on system requirements.

Depending on materials of the protective layer 7122, 8122 or 9122 andthe counter electrode 7104, 8104 or 9104, the protective layer 7122,8122 or 9122 and the counter electrode 7104, 8104 or 9104 can,respectively, be the anode and the cathode, or vice versa. Toillustrate, as an example, an active material of the counter electrode104 is silver (Ag); however, the present disclosure is not limitedthereto. However, silver, when exposed to the air, tends to react withoxygen and water vapor and becomes silver oxide, wherein an oxidationequation can be expressed as 4Ag+O₂→2Ag₂O, and wherein a standardreduction potential of silver/silver oxide is 1.17 V. When silver isoxidized into silver oxide after being exposed to the air, it willresult in toxification on a surface of the counter electrode 7104, 8104or 9104. In this case, the capability of the counter electrode 7104,8104 or 9104 to receive or release additional electrons will bedecreased, and therefore, the bottleneck effect between the workingelectrode 7102, 8102 or 9102 and the counter electrode 7104, 8104 or9104 cannot be effectively mitigated.

As shown in FIGS. 22 to 24 , the implantable biochemical test chip 7000,8000 or 9000 is disposed with the protective layer 7122, 8122 or 9122,and the protective layer 7122, 8122 or 9122 is electrically connected tothe counter electrode 7104, 8104 or 9104. In some embodiments, theprotective layer 7122, 8122 or 9122 is configured to protect the activematerial of the counter electrode 7104, 8104 or 9104. In someembodiments, the protective layer 7122, 8122 or 9122 can includestannous oxide (SnO). Stannous oxide, when exposed to the air, tends toundergo oxidation reaction with water vapor, wherein the reaction can beexpressed as SnO+H₂O→SnO₂+2H⁺+2e⁻. A standard reduction potential ofsilver oxide/silver is 1.17 V, whereas a standard reduction potential ofstannous oxide/tin oxide −0.09 V. Hence, in the present embodiment, theactive material of the counter electrode 7104, 8104 or 9104 is thecathode, and the protective layer 7122, 8122 or 9122 is the anode. Whenthe implantable biochemical test chip 7000, 8000 or 9000 is exposed toan environment with water vapor, the potential difference (E_(cell) ⁰)is 1.08 V. Because the potential difference (E_(cell) ⁰) between the twois greater than 0, a change in free energy is smaller than 0, and hence,a following reaction will occur spontaneously: Ag₂O+SnO→2Ag+SnO₂. Inthis case, a half-reaction taking place on the active material of thecounter electrode 104 is Ag₂O+2H⁺+2e⁻ →2Ag+H₂O.

Therefore, silver oxide in the counter electrode 7104, 8104 or 9104 isreduced to silver as a result of the oxidation reaction of the stannousoxide in the protective layer 7122, 8122 or 9122. When the protectivelayer 7122, 8122 or 9122 undergoes oxidation reaction, the activematerial of the counter electrode 7104, 8104 or 9104 undergoes thereduction reaction, thereby slowing the oxidation reaction caused byoxygen and water vapor in the air. Further, through the oxidationreaction between the water vapor in the air and the stannous oxide, theactive material of the counter electrode 7104, 8104 or 9104 is furtherprotected, so as to keep the active material of the counter electrode7104, 8104 or 9104 stable. Therefore, by disposing the protective layer7122, 8122 or 9122 in the implantable biochemical test chip 7000, 8000or 9000, the active material of the counter electrode 7104, 8104 or 9104can be prevented from being damaged before a specimen measurement isperformed. Compositions and materials of the active materials of thecounter electrode 7104, 8104 or 9104 and the protective layer 7122, 8122or 9122 are not limited to those described above. In some embodiments,the compositions and the materials of the active materials of thecounter electrode 7104, 8104 or 9104 and the protective layer 7122, 8122or 9122 are chosen so that the potential difference (E_(cell) ⁰) betweenthe two is greater than 0. In some embodiments, an absolute value of thepotential difference (E_(cell) ⁰) is greater than 0.

The present embodiment provides a protective layer 7122, 8122 or 9122 inthe implantable biochemical test chip 7000, 8000 or 9000. The protectivelayer 7122, 8122 or 9122 can maintain stability of the active materialin the counter electrode 7104, 8104 or 9104, so as to protect theimplantable biochemical test chip 7000, 8000 or 9000 and mitigateunexpected damage to the implantable biochemical test chip 7000, 8000 or9000 caused by reaction with an environment before the implantablebiochemical test chip 7000, 8000 or 9000 is used in a specimenmeasurement, thereby maintaining or protecting a capability of thecounter electrode 7104, 8104 or 9104 of the implantable biochemical testchip 7000, 8000 or 9000 to receive or release additional electrons.

FIG. 25 is a chart showing signals detected on a counter electrodeaccording to the present embodiment and a counter electrode of acomparative example under different concentrations. In detail, data Cand data D show the signals detected on the counter electrode 2104,3104, 4104, 5104, 6104, 7104, 8104 or 9104 according to the presentdisclosure, while data A and data B show the signals detected on thecounter electrode according to the comparative example. As shown in FIG.25 , at a low sugar concentration, there is a smaller difference betweenthe signals of the counter electrode of the present embodiments and thecounter electrode of the comparative example. However, when the sugarconcentration is increased, the counter electrode of the comparativeexample exhibits a bottleneck.

FIG. 26 is a chart showing signals detected on an electrochemical systemaccording to the present embodiment and an electrochemical system of acomparative example over time. FIG. 26 may represent a long-termstability test of the counter electrode of the present embodiment andthe counter electrode of the comparative example. The implantablebiochemical test chip of the electrochemical system 1000 and animplantable biochemical test chip of the electrochemical system of thecomparative example are implanted or inserted into a same chemical tankwith a fixed concentration. The reaction signals are continuouslydetected. In detail, data M shows the signals from the electrochemicalsystem 1000 according to the present embodiment, while data N show thesignals from the electrochemical system according to the comparativeexample. As shown in FIG. 26 , the early performance of theelectrochemical system of the comparative example is consistent with theelectrochemical system 1000 of the present embodiment. However, on the15^(th) or the 16^(th) day (see point Y), a bottleneck effect occurs tothe counter electrode of the electrochemical system of the comparativeexample due to disintegration of a conductive medium on the counterelectrode. On the other hand, the electrochemical system 1000 of thepresent embodiment has better stability because of its own redoxability.

Although the disclosure and its advantages have been described indetail, it should be understood that various modifications,substitutions and replacements can be made without departing from thespirit and scope of the present disclosure as defined by the appendedclaims. In addition, the scope of the present application is not limitedto specific examples of processes, machines, manufactures, materialcomponents, means, methods and procedures described in thespecification. Those skilled in the art can understand from thedisclosure of the present application that existing or future developedprocesses, machinery, manufacturing, and materials that have the samefunctions or achieve substantially the same results as the correspondingembodiments described herein can be used according to this disclosure.Accordingly, such process, machine, manufacture, material composition,means, method, or step fall within the protection scope of the presentapplication.

What is claimed is:
 1. An electrochemical system, comprising: anelectrode unit, comprising a working electrode and a counter electrode,wherein a current density of the counter electrode is greater than acurrent density of the working electrode; and a reactive unitelectrically coupled to the electrode unit.
 2. The electrochemicalsystem of claim 1, wherein the current density of the counter electrodeis greater than or equal to twice the current density of the workingelectrode.
 3. The electrochemical system of claim 1, wherein an area ofthe counter electrode is smaller than or equal to an area of the workingelectrode.
 4. The electrochemical system of claim 1, wherein the counterelectrode is a cathode, and a standard reduction potential of an activematerial of the counter electrode satisfies E_(s) ⁰>E_(m) ⁰−E_(v) whereE_(s) ⁰ is a standard reduction potential of the active material, E_(m)⁰ is a standard reduction potential for a concentration reaction on theworking electrode, and E_(v) is a potential applied by a measuringapparatus when providing a measuring reaction.
 5. The electrochemicalsystem of claim 1, wherein the counter electrode is an anode, and astandard reduction potential of an active material of the counterelectrode satisfies E_(s) ⁰<E_(m) ⁰−E_(v) wherein E_(s) ⁰ is a standardreduction potential of the active material, E_(m) ⁰ is a standardreduction potential for a concentration reaction on the workingelectrode, and E_(v) is a potential applied by a measuring apparatuswhen providing a measuring reaction.
 6. The electrochemical system ofclaim 1, wherein the reactive unit and a target analyte undergo aprimary reaction, and the counter electrode is configured to undergo asecondary reaction, wherein the secondary reaction does not interferewith the primary reaction, and the secondary reaction allows the counterelectrode to receive or release additional electrons.
 7. Theelectrochemical system of claim 1, further comprising: a protective unitelectrically coupled to the electrode unit, wherein the protective unitis configured to oxidate the electrode unit after the electrode unitreceives an electron or to reduce the electrode unit after the electrodeunit loses an electron, wherein there is a potential difference(E_(cell) ⁰) between the protective unit and the electrode unit.
 8. Theelectrochemical system of claim 7, wherein the potential difference(E_(cell) ⁰) is greater than
 0. 9. An implantable biochemical test chip,comprising: a substrate; a biocompatible coating over the substrate; anelectrode unit, between the substrate and the biocompatible coating,wherein the electrode unit comprises a working electrode and a counterelectrode, wherein the counter electrode is configured to receive orrelease additional electrons through a self-secondary redox reaction,and a current density of the counter electrode is greater than a currentdensity of the working electrode; and a reactive layer electricallyconnected to the electrode unit.
 10. The implantable biochemical testchip of claim 9, further comprising: a first end connected to ameasuring apparatus and a second end implanted into a specimen.
 11. Theimplantable biochemical test chip of claim 10, wherein the second end iscovered by the biocompatible coating.
 12. The implantable biochemicaltest chip of claim 9, wherein the reactive layer is between the workingelectrode and the counter electrode.
 13. The implantable biochemicaltest chip of claim 9, wherein the reactive layer covers the workingelectrode or the counter electrode.
 14. The implantable biochemical testchip of claim 9, wherein the substrate has an elongated structure, andthe biocompatible coating surrounds the substrate.
 15. The implantablebiochemical test chip of claim 9, wherein the electrode unit furthercomprises a spare electrode over the substrate.
 16. An implantablebiochemical test chip, comprising: a substrate having a measuring endand an implantable end; a biocompatible coating over the substrate; anelectrode unit, between the substrate and the biocompatible coating,wherein the electrode unit comprises a working electrode and a counterelectrode, wherein the counter electrode includes an active material;and a protective layer, electrically connected to the electrode unit,configured to stabilize the active material of the counter electrode.17. The implantable biochemical test chip of claim 16, wherein theprotective layer is located at the measuring end.
 18. The implantablebiochemical test chip of claim 16, wherein the protective layer isarranged adjacent to the counter electrode.
 19. The implantablebiochemical test chip of claim 16, wherein there is a potentialdifference (E_(cell) ⁰) between the protective layer and the electrodeunit.
 20. The implantable biochemical test chip of claim 19, wherein thepotential difference (E_(cell) ⁰) is greater than 0.